CA2075050C - Specificity and convenience of the polymerase chain reaction - Google Patents

Abstract

Improvements to the polymerase chain reaction (PCR), a process for in vitro enzymatic amplification of specific nucleic acid sequences, can be achieved by changing the way that PCR reagents are mixed and the enzymatic reaction is started and by the replacement of mineral oil, commonly used as a vapor barrier to minimize solvent evaporation, by a grease or wax. The use of such mixtures allows for the delay of reagent mixing until the first heating step of a PCR amplification, thereby reducing the enzymatic generation of nonspecific products which occurs when a complete mixture of PCR reagents, with or without test sample, stands at room temperature or below. These mixtures increase the shelf-life of PCR reagents and increase protection of the laboratory environment against contamination by PCR product.

Description

~ ~ 7 ~ 0 5 ~ ' Il\IPROVEMENTS IN THE SPECIFICITY AND CONVENIENCE
OF THE POLYMERASE CHAIN REACTION
Field of the Invention The present invention describes novel compositions and methods for simplifying and improving the specificity of the polymerase chain reaction, a procedure for amplifying specific nucleic acid sequences which finds broad use in the fields of genetics, molecular 0 biology, cellular biology, analytical biochemistry, clinical chemistry, and forensic science.

Back~round of the Invention The polymerase chain reaction (PCR) is a chemical method of increasing by many orders of m~gnitllde the concentration of a specific nucleic acid sequence in a test sample.
The PCR process is disclosed in U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188.
In PCR, a test sample believed to contain one or more targeted nucleic acid sequences is combined in a total volume of usually about 20-200 ,ul with the following reagents: an aqueous buffer, pH 8-9 at room temperature, usually also cont:~ining approximately 0.05 M KCl; all four common nucleoside triphosphates (e.g., for DNA
polymerase, the four common dNTPs: dATP, dTTP, dCTP, and dGTP) at concentrationsof approximately 10-5 M to 10-3 M; a magnesium compound, usually MgC12, usually at a concentration of about 1 to 5 mM; a polynucleotide polymerase, preferably a thermostable DNA polymerase, most preferably the DNA polymerase I from Thermus aquaticus (~
polymerase, subject of U.S. Patent No. 4,889,818), usually at a concentration of 10-1~ M to 2 5 10-8 M; and single-stranded oligonucleotide primers, usually 15 to 30 nucleotides long and usually composed of deoxyribonucleotides, cont~ining base sequences which are Watson-Crick complementary to sequences on both strands of the target nucleic acid sequence(s).
Each primer usually is present at a concentration of 10-7 M to 10-5 M; primers are synthesized by solid-phase methods well known in the art of nucleic acid chemistry.

3 o In the simplest form, PCR requires two primers for each target sequence. These primers, when annealed to the opposing target strands, have their 3 ' ends directed toward one another's hybridization sites and separated by about 100 to 1,000 nucleotides (occasionally up to about 10,000 nucleotides). The polymerase catalyzes magnesium-dependent, template-directed extension of each primer from the 3' end of the primer, 3 5 incorporating nucleoside monophosphates into the growing nucleic acid and releasing pyrophosphate.

WO 91/12342 PCr/US91/010?' This extrnsion reaction CO~ CS until the poly~lase reaches the 5' end of the trmplste strand to which the e, l~ n~ d primer was ~nn~ ed, at which point the poly,l~.~ is free to bind to another primer-t~npl~tP duplex and catalyze ext- nSion of that primer rnolç~lllç; the e~ nC;on ,~&;lion also stops if the reaction ~ ule is heated 5 to t~ ,s sllffiriçnt to separate the template from the extPn-1~1 primer before the el~y,l~ has l~&ched the S' end of the tçmplste~ After the en~y,l,e has worked long enough to transform a large fraction of the primer-template duplexes into double-stranded nucleic acid, the latter can be denatured at high tell~la~ , usually 90 to lOO-C, to create two single-~ n~cd poly..~,ck~ es, which, after cooling to a 10 t~ ; where they can be annealed to new primer mr)~ lçs, serve as tçmpl~tes for another round of w~y~LIe catalyzed primer e,~t. ..~ ~n Recal~ce both DNA strands serve as te...~ tç each round of nucleic acid reE!lirsti~n al)pl~. ;...~lçly ~oubles the conce-n~ Lion of the specific nucleic acid s~vçnre defined at its ends by the two primer s~uçnr~s Th~ ,ful~i, the total ~n~.5..~.alion incl~ in the target nucleic acid 15 s~uçnre in a PCR ~mpli r~rAI ;on is by a factor of applr~ tely 2n, where n is the num~l of completed thermal cycles ~l~,. cell â high h- .~ C where double-stranded DNA is denalul~l and a lower l~lll~l~u~ or set of Llll~latul~s (40 to 75 C) where primer Llll~lalc ~nne~ling and primer ~ - t~ ~~;OI- occur.
~hho~l~h one can move PCR reaction tubes manll~lly back and forth ~l~n~n 20 ll~e ,~ . A baths in the two ~ Jf ~ ranges, PCR most co,l"llollly is peI rollllcd in an ~ulO~.~?t~ m~la~u,c-controlled ..~c!-ine, known as a "them~al cycler," in which a fi~ cessûl is ~lu~a ll,lled to change the t~ .nl)f ~t-..c of a heat-eYcll~ngt- block or bath cc~ g l~ i~l tubes back and forth among several spe~firA ~m~ldlulcs for a sre~-ifi~d nulll~l of cycles, holding at each ~ , for a spe~fi~ time, usually 25 on the order of one-half to two ...;~ t~,s Such a thermal cycler is colll~ ,ially available from Perkin Elmer Cetus Insl"lm~ . The total cycle time is usually less than 10 ...;.--lhs, and the total l~ bcr of cycles is usually less than 40, so that a single, multi-cycle ~nplifir~tion, amplifying the targeted nucleic acid s~u~,ncc 105 to 101~
times, nonn~lly takes less than seven hours and often less than four hours.
The practical benefits of PCR nucleic acid ~nlrlifir~tion have been rapidly appreciated in the fields of gçnetics, ms)le~ul~r biology, cellular biology, clinical chcl"islly, forensic s~ence7 and analytical bioch.omictry, as ~les~ibe~l in the following review volumes and articles: Erlich (ed.), 1989, ~ Technolo~y. Stockton Press (New York); Erlich et ~1. (eds ), 1989, Polym~r ~h~ Reaction. Cold Spring Harbor Press (Cold Spring Harbor, New York); Innis et al., 1990, PCR Protocols~
Ac~len-ic Press (New York); and White et al., 1989, Trends in Genetics ~:185-189PCR can replace a large fraction of ~lecular cloning and mutagenesis ~ l~OnS

O 91/12342 PC~r/US91/01039 3 2~75050 cc,.. ~nly ~IÇulllled in bacteria, having advantages of speed, ~impli~ity, lower cost, and S-~ f-l;n'Y in-,l~ safety. ~ Jne~ PCR pennits the rapid and highly sensitive qualitative and even 4..~ e analysis of nucleic acid sequ.,nces, often with greatly ~ ased safety be~v~ so much PCR product is made that no~i~ot~pic 5 detection modes suffice.
Despite rapid and broad adoption of PCR by a range of biol~ral and çhPmi~l ;n-,s, PCR still ~-~s several practical limitations that must be ove,~cullle forfull re~li7Ati- n of the analytical and S.~ ,tiC potentials of the process. Some of these l;...;lA1;f nc are '1;~US~ in turn, below.
Many ~ ~pl;rir l ;- n~ yield l-- n~,f.-; r.c side ~,l~lu ils dirr~,~g in size and sc~lu~ nr~ from the sc~q"en~e Id~ by the primers used. So...- -I;...f-S nrnspecifi~ity is caused by mis-p. ;...ing, where primers have been ~nne~led to non-target se~lu~,nces, also present in the nucleic acid of the test sample similar to the target sequence ~lthough the genomic DNA c~-.. -~nly con~-;n~ in PCR test s~les has ~ h~ ;ly 15 been t~wuE;hl to be completely double-~ n~ c~lu~s used to prepare DNA forAn~l-lirir~ n appear to render that DNA, to a large extent, single-stranded. Single-d DNA is esper~ y ~-~epl;~le to mis-p.;~ g if mixed with a comrlet~ set of PCR l~agellb at ambient or sub-ambient ~ ,s. Many PCR l~&i~ns also yield primer dimers or olignm~s, ~ouh~ h~n~led side pl~lU~ki COl~ ning the se~uennes; of 20 several primer nle ~lles joined end-to-end, the yield of which correlates negatively with the yield of A~ ;r~l target s~u~nce. "Low-copy-number" PCR, ~h~ the total num~l of initial target se~uen~s is less than about l,000, is espe~lly prone to primer .~ c!n and mi~ g, which reduce spe~ifir pn,llucl yield, yield precision, and ~ nplific2tiQn S~ ;r..~
The high amplification factor and res~llting high sensitivity of PCR renders theprocess e-sre~lly v!lln~ ~le to back contam nation, where ~mplifi~ target from one .i~n iS ~r~tlrnt~lly 1- ~ r ~ ~~,d into a subs~u~nl reaction using the same primers and gives a fal~-positive result in the later rea~on In principle, PCR could be ~lÇulllled ~veral times faster than current practice allows, being rate limited in part by the speed of ~lll~ldlUl~i change during thermal cycling. Clinical ~lia~os1ir applications of PCR would espe~ ly benefit from total nrlifir~tion times of 30 to 60 .~ t~-S instead of ~veral hours.
Lower PCR costs and incl~ased speed and precision could be obl ~in~A if the l~agen~ could be milced in large batches, aliquoted into the small l~au~-ol tubes (usually one-half ml total capacity c~ t~ ;ng 20 to 200 ~1), and stored for long periods ~l~n plGp~ualion and use will.uul loss of ~.nrlifir~tir.n effiriency.

WO 91/12342 PCl/US91/010~

The h~ ,t~role standard PCR art has called for covering the rqueo~l~ reaction ul~i with 50 to 100 ~1 of mineral oil to prevent solvent c ~ ir,n during the several hours of hPrting The mineIal oil overlay il~ll~luces several practical problems:
(a) mineral oil co. .~ -- . .;n -~ ;on of ~ n ~ samples withdrawn for post-PCR
analysis, often ~ ;nE extraction with ha~.lous water-;..... ;~cible organic solvents to avoid int~.fi .~nce with post-PCR pl~es~;ng; (b) a l~ l;r~n of thermal equilibration during the~mal cycling (because of the cig,.;r.~ heat cap~ity of the oil layer), ul.;l~ing the total cyclc time; and (c) o~ q~ ~lu~l;on from some batches of mineral oil of ;~ s which appear to inhibit PCR, nP~es~ .l;ng rigorous 10 quality control of n~ineial oil.
The present invention significandy miti~qt~s he l;n-;l~ of PCR ~liccl~csed above, by several s.l,~ .ingly simple n~;r.~ ~;on~ of PCR pr~tice and m~teri~l~
~ecdu~ primer dimer and olignrn~r fcN~lion can occur ~hcn~ ~. all of the PCR
~Ib are mixed, even at ambient and sub-ambient temporatules in the ~bsence of 15 thermal cycling and in the r~sPnr~ of target DNA, ~ dion of at least one reagent from the others in a way such that all l~agen~ do not come ~og,~ before the first q.~rlifir~qti(~n cycle can reduce prim~ olieO~ ;0,. and, in doing so, can gready extendtheshelf-lifeoftheincompletereagentmixturewithoutgreadyco...plir~l;.,gfinal re~tion set-up. Such se~,~alion also can ...:-.;...;-., mis-rmin~ during the poorly 20 controlled interval over which PCR l~agenls and test sample C~ ;1y are mixed and stored at ambient or subambient t~.ll~dul~S before dhe start of thermal cycling,esperqlly if se~,gat~d lcag~ and test sample are inll~luced into the PCR reaction tube with minimql mixing.
Several çl-~ r~l pl~lies of ma~-~ . confer special advantage to se~Ea~,llg dhe m~.. ~,~;.. colll~u"d from the odher PCR l~agel,t~ (as opposed toSe~E,aling ellL~llle~ , or dNTPs) when setting up a PCR amplification. Fattyacid salts of ma~;..e~;.--.. are potentially soluble in oil, grease, or wax, yet also ially water extractable when the organic layer is C41~ d with the hot aqueous l~g~,nts during PCR. That way reagent se~,E;a~ion and reaction tube preparation can 30 be simplifi~ by inc~l~atio" of the mq~ ... into the organic layer rather thanpl~palali~n of a sep~ e aqueous reagent which must be added. Being inorganic, mag..~s;~ salts need not be pl~a,~,d and stored with special p~caul;on~ against mic~bial CQ.~ ;n~l;On, a c~ u- pl,~lçm with llfL~lul.,s conl~ l-g uucleos;~1e 1 . ;pho~hales, ~ "~ e, or l,li,l~l~. The phos~.h~ Gs and yho~l.ho l;estf rases which degrade ,~,cl~:~le ~ h~s~k~ and primers often are ma~.. ~;.. -r~u;. ;ng, so that storage of the biological reagents without ma~l.f ~;---.. (possibly also with a trace of che~ r to bind any small amount of m~gn~s;u... present) improves shelf life and ~ 91/12342 PC~r/US91/01039 _ 5 2075050 ct~nre to cQr.~ ;on by enLy~es or by l~ ubcs which secrete the enzymes.
Segregation of any potassium salt with the m~..~ .. cc~ uw~d and away from the protein and nucleic acid also im~lu.es resi~t~nre to microbial col-c~ ~ion of reagents, b~cause l~O!~c~;.. ion also is needed for cell growth.
The present invention provides an es~peri~lly effective mode of reagent se~G~Iion by providing means to replace the mineral oil overlay with a layer of grease or wax, the solidity of which at room ~ or below creates a barrier against mixing of aqueous reagents se~.,gà~ above and below the grease or wax layer.
Thermal cycling tums the solid barrier into a lighter-than-water liquid of low viscosity, which is ~ e d by an ~ queou~C, layer above; the upper ~queollc layer con~ C allPCR reagents not present in the lower ~u~us layer. Cun~;luenlly~ reagents previously sc~ga~ed mix to create a complete l~lioll with the help of the c~n~i~er~le thermal convection which ilc~c,...y -i~-s heating of the reaction tube. The melted grease or wax creates a vapor barrier to ...;n;..~ , solvent eva~lalion during 15 thermal cycling and, upon cooling after ~nplifir~tion is conlrlete, re-fonns a solid barrier which, among other things, reduces the ease of PCR p~lucl dispersal into the envir~"~nl when reaction tubes are opened, thereby re~ucing the likelihood of back-C~ 15 1 I;n~l ;ng later re~ tionC-~
A photo st~riti7~tinn process to prevent back~4..~ ;nn has been developed 20 and involves the i~ r~;Qn of ps~ n and is~l)sr,.AtPn derivatives to photo st~riliPCR p~lucl in a way which permits post-PCR analysis but ~ er,b use of that product as a te-ntpl~te in subs~uent ~nlrlifieations. However, the psoralen and isol~ulalen photorea~,nt~, c~.. l~nly added before ~rnrlifir~ti--n, appear occ-c:on~lly to inhibit PCR. I~ul~ e, the 111~"1~ ion I~UU~d for PCR is likely to reduce 25 the affinity of l)ho~lcagent for double-sl-; n~l~l nucleic acid (Hyde and Hearst, 1978, Bioc}.~ 17:1251-1257), thereby reclurirlg l,ho~lcagent efficiency or increasing greatlythephotoreactantcv..~e~ l;nnl~uil~dforpracticalphoto-stP.rili7~ti~n The repl.~re..~ ~--t of mineral oil with grease or wax, as provided by the present invention, permits a pl 7~tir~1 moflifirAti-n of the photo s ~ili7~tiQn ~ ul~ that l lcie~
30 int~.r~..,nce of the reagent with ~-nrlifir~tinn of new target nucleic acid and should inc~ease photoreaction effirienry. After ~n~rlifir~ti-~n in the absence of ~holc,~agent and after re-soli-lifir~tion of the grease or wax, the reaction tube can be opened without fear of conlA...;.~ ;..g the en~,~"lll~,nl with PCR IJlu~lu~;L An aqueous s~olllti~n of pholc~lcagent and a chelating reagent which binds m~..es;....~ can be placed on top of 35 the grease or wax. Closure of the tube and a simple brief heating step to melt the grease or wax allows mixing of photc"~,agent and chel~t~r with PCR product; this Illi~CIUl~:; iS

WO 91/12342 PCI/US91/010~~
207SI~50 6 now ready for optimal photo-sterili7~tion~ as the chel~tion of m~ .... ion allows tight binding of ~holol~ a~nl to nucleic acid.
After PCR A~ul.lir~r-l;c)n, cc~ sn practice is to detect r nplified nucleic acid by reachng the amplified nucleic acid with a reagent that caTries an analytical signal 5 gell~.~ol or a reagent that fn~ilit~t~os separation of ~nlrlifi~d nucleic acid f~m other co~ onenls of the PCR reaction ~ . Such reagents are ~lesign~A to bind very tightly to ~ "l.l;r.kd nucleic acid, eitherb~ e they include nligonllcleotides with se~ 4s co.u~ ,t ~ to part of the target se~u~ e (nucleic acid probes) or be~ e they bind to m~le ~ -s. such as fluol~ scein and biotin, which are conveniently 10 ~ .hr~ to primers or the ,~ e I. ;l~hos~kAIes incol~vl~t~d into PCR product.
Signal ~ g ~.~b~ es that might be ;~ ,ded in such det~tiQn reagents conlrri~e r~ ico~s nuol~holus,chemilll~ esce~tmoieties, c~ loc-he.~ -escent catalysts, and catalysts in general, such as enzymes. Se~al~on-l)lv~ g substances comprise antibodies, avidin, streptavidin, biotin, high-affinity haptens like fluorescein, magnetiC particles, denser-than-water panicles, latices capable of ~g~,lul;n~ n and adsoll~nls capable of binding to either single-st~n~1~ or double-stranded DNA or to specific nucleic acid se~uences.
Such ~let~ction l.,agenls often are h~COn~1~AI;hle with PCR ~mplifin~tion~ either bec~nse, like most proteins, they are inactivated by the prolc ng~d heating in PCR or 20 b~ e, like most separation-p~v~ in~ sub~ ce5, they might inhibit PCR by removing lca~.,b fTom sol~tior . Thel~ef~.l." it is generally be-n~.fici~l to add PCR
product ~let~ti~n reagents after amplification has been completed or almost c~mple As in the case of photoste~ 7qtic-n~ the present u~ ion allows such late addition to the PCR ~ ion tube to occur with minim~l risk of coul; ,~;nql ;.-g the labola~l~ with 25 ~rnrlified nucleic acid, ber ~u~ PCR product can be sealed beneath a layer of grease or wax.
Still another situation in which late aA~liti~n to a PCR reaction is desi~ble col-ce. I,.c "nested primers," wh~ ;ln PCR spe~ifi~ity is enh~ eA by following an initial rc,~l;on vith an ~nlrlifil~tion using primers comple ~ y to sec~u.,nces not 30 present in the original primers or primer-complc----~ --l~- ~ regions but ~ l;r.rA. by ext~.n~ion of the original prirners. The present invention allows late ~ 1itio~ of the internal primer pair of a nested primer system with much reduced cr nç~ about cc l-~z ..;n~ g the labol~Lc,l y en~in;>,.ll,cn~ with amplified nucleic acid. After such litinn, only one or a few arnrlificatinn cycles are needed to ge.l~l~L~ enough of the 35 shorter PCR product to detect.
Many other citll~tionC exist in which late addition of a substance to a PCR
amplification has benefi~i~l effects on PCR sensitivity, s~ir,ciLy, conveni~nce and '~ 91/12342 PCI /US91/01039 7 20750S0~:
product analysis. In every case, the present invention advances the art by allowing that addition to occur (a) under cor-lhi~ n~ where qnlrlifi~d nucleic acid is sequestered;
and/or (b) at elevated ~,n~.~lu,c.

S..n....n~ of th~ Tnv~ntinn In a first aspect, the invention is a method of in.,~ g the s~ y of PCR
amplification and of in~ asing the shelf life of pre-mixed PCR req.~nt~, wh~ PCR._agerlb (ecc~ ling the test sample co~ln~ .g the target DNA) are f.. ~ q-ted as at least two non-ove~lapph~g subsets which can be stored for long periods of time (many months) will~oul reaction or degr~d-q-tio~) being b~ught together in a PCR reaction tube 10 with minimql mixing shortly before (within a half hour of) thermal cycling As~les~ribe~l more fully below, this s~c~lion can be a~l,ie.od by (1) placing all but one (or more) ess~o-nti-ql PCR co. ..pon~ nt in one conl~;n~ . and the other essentiql cc"l,~ner,t(s) in another cou~;-.c, (2) by placing all e~nSiql PCR reagents in a single con~;.inf r but having one or more esse-~ 1 reagents sequest~-red from the other esse .~
15 re-q-~nt~ This latter se~lue~llalion can be achie~ed by merely placing a barrier, such as a wax layer, ~l~n the reagent l~lul~,s or by e...he~d(l;..g one or more essenti-q-l reagents in a matrix or gel, such as an agarose or acrylamide gel, or even a liposome.
In a pl~f~l~d ell,bod;.~ 1, all reagents except a m;Ag..~ ... co~ ou,ld are mixed in advance of amplifiration, and the complete ,~ilion ll~lu,c is plC~ d in a 20 way which ,..;n;...i,~s mixing of the m~ e~iv~ CC.n~n(l with the ,~ ;ni-~g reagents until the first amplification cycle is begun. Spe~ifir~lly~ this ~ ...k)~l;...~ ~-l conlrrice~C the layering of liquid sol~ltion-~ or ~ nC of the ma~-rC;~ co...l o~i-d the m~ ll-free lG~ge~ , and the test sample in the l~lion tube, preferably in a way such that the test sample or a layer of solvent lies ~t~n the two reagent form~ tions.
25 Preferably any pot~csillm salt inrl~lde~d in the PCR ,eacl~n ~i~lulC is f~ rmnlAt~ with the m~gnes;v~ lpound, not with the other re~nts.
In a second aspect, the invention co.--~.. i~s co,l,posilions and methods which simplify the se~ alion of any subset of PCR reagents from the compl~ .n~. y subset, whcl~,;n each subset is formlllAte~ in an ~ucous ~ ;on or solution and a30 layer of grease or wax is placed b.,lv~~n the two subsets before amplific~tion. The grease or wax melts into a lighter-than-water liquid of low viscosity during the first ~mrlifir~tic)n cycle, wl,e.c.-pol~ mutual ~ e...h.ln of the melted grease or wax and the aqueous layer above it and convective mixing of the now united aqueous reagents permits; mrli r.c"~;O~.
35In a third aspect, the invention coll""ises col.~l os;l;on~ and methods which simplify the segregation of any subset of PCR reagents from the complc...f-nt~.y WO 91/12342 PCI/US91/010~
~o~Q50- 8 sub~t, whel~;;n one sub~t is hl~at~xl into a lighter-than-water oil, grease, or wax which is layered on top of any ~1ueo~ls ~ c:on or solution of another subset.
H.o~ting during the first amplific~tion cycle melts any grease or wax into a liquid, and heating of the t 1ueol.c layer results in extraction of the miccing reagent subset from the 5 lighter-than-water overlayer into the ~.I.J~ s layer, convective mixing of the now united reagents pelmits amplir~c ~ n One specific emk~iim~nt of this aspect of the invention consists of an e-mvl~iQn of an aqueous solYtion or suspension of a subset of PCR l~ag~lls in the oil, grea~, or wax. Another e-nk~3;~ n~ is a solution of thesub~t of PCR l,,agel.ls in the ~il, grease, or wax. Spe~ifi~lly ~ f~ d PCR reagent 10 fr~rm~ tinn$ for .liccQll.tir.r~ in the oiL grea~, or wax are ma~ne~;~.... fatty acid salts, and t~ yl--n..)o~ n salts of the n~clP4s:~1e ~ .osph~s and of the prim.ors, In a fourth aspect, the invention co..q.. ;~s co..~ .s which improve the function of the second aspect of the invention by cll~nging the physical l~lup~ ies of the grea~ or wax. Specific ~...~l;...~nl~ CQ~ ;~; (1) a sollltion of ~v~ r~ n~ in the 15 grea~ or wax; (2) a 50l~tion of s~ r~ n~ in the ~que~ouc layer below the grease or wax; (3) an ~rlifir~tion co~ , the inner surface of which is h~hilic; (4) a ~u~n-:ol~ of plastic particles in the grease or wax; and (5) a layer of plastic mesh sVspenAeA in the grease or wax. ~efell~,d amplification co~ nc ~ with hydl~hilicsllrr3q~s are plastic tubes which have been (a) coated with a s~ rq ~!iq~nl, (b) plasma etched in an oYi~li7ing en~ilvn.. l, (c) treated with a str~ngly oXi~li7ing liquid, or (d) cast from a resin melt to which ~,u- r~clAnt has been added.
In a fifth aspect, the invention co~nrri~es any cm~lAi~le~ for ~lÇ~,lmi,lg an aqllf al~ cl~ r~l reaction which CQ~ ~;ct~ of a vessel and an amount of a wax sufficient to cover completely the ~d su~face of the ~queo~l~ COI~lf nl~ of the vessel, wherein 25 the inner surface of the vessel is hy~hilic or the waY. has been mixed with a nonionic sllrr~q~t~-lt In a siYth aspect, the invention cc~mr~es kits for PCR amplifir~q-ti~n of nucleic acids. These kits c~ pllse the novel f~rmlll~ti~ns of the PCR compositions of the present invention and can also conlrri~ instructions for carrying out PCR with the 30 cc~m~o~ilions of the invention.

D~t~il.oA Descru~ti-)n of the Invention The first four aspects of the invention improve the spe~ifi~ity of PCR
amplifi~tion by ~ enLing any catalytic reaction of nucleic acid polymerase with other reagents until the first ~nnrlific~tion cycle. In particular, the foll,lalion of PCR side 35 products known as primer oligom~rs, a reaction which occurs even at room te~ )e,~ture and below in the ~bse-n~e of nucleic acid template, is greatly reduced. Also disfavored ') 91/12342 PCI/US91/01039 is the mis-primed amplifir~ n of ~ .sl~;r~r targets when the test sample cont~in~
single-~ll~ ded DNA. Amplification of target PCR products often is increased and,~,nd~ ,d more reproducible when side products are ~u~ ~s&~ The increased reaction specificity res~lting from the invention often results in ~ b~ f~ lly pure ~mrlified 5 target sequence, gready simplifying PCR plUllU(;l analysis and 4..~f.l;l;.t;on For eY~mrlt~, PCR product can be i~1el~;r~&~l c4..r.Af-~.lly on the basis of ek~llophoretic or ch,~ ~ ~hic migration rate widlout resorting to slow, laborious, relatively u~ e At~tç~tion modes like nucleic acid probing. In particular, fast and highly ~-~,n~ l HPLC analysis of PCR IJl~lu~;l now is ~ ;t ~l The first four aspects of the invention also s ~ the buL~ m~nllfP~lre of ready-t~use PCR reagent formulations with shelf lives on the time scale of a week or more. P~ U1~ of all l&~,~ t'nt~rally results in a shelf life of no more than a few days, ap~ ly bc~a ~e of side reactions such as primer oligomt.r f~rm~ tion~ which occur at room te~ A ~ or below in the absence of ternr~ nucleic acid. As soon as15 at least one reagent is se~gdt~,d from the others until the first ~rnrlifi-~tiQn cycle, these side reactions cannot occur. Bulk .~ r~v~ of ready-to-use reagent fnrm~ tion~
incl~s the speed, co~ en;& nc~ ~ and reliability of PCR by relieving dhe user of dhe need to make careful lllL~IIU. s of l~gell~ at time of use. Often, time-of-use formlll~ion entails ixing of small volumes and the pl~ala~ion of just a few reaction 20 mib~ ,S at a time, reA~cin~ the intra-day and inter-day An~ r~r~ation precision.
The mag~P ~;... - - se~igalion (preferably also co. ~ ~1" ;c; ~-g ~!; c~ n segregation) of the first aspect of the invention is an impro~ ~nl over the seE,I~ lion of other PCR
r&~g~nt~, such as CI~LYI1~, plilll.,~, or nuclPoside !~ hosph~t& s for seve~l reasons. As the only non-biological, non-biodegradable, PCR reagent, the m~.Ps;.. co"l~)ound 25 l~UJl-,S the least stnng~nt (e.g., sterile) forml~l~t-ion and storage. Preferably, the other ~g~,nls are fi~ ~g~ ,r and stored in a manner which l~ s their biodegradability and greater ~ n ~l lability. FY~lllci~n of an essc-lt;fll ,nic,obial l-;ent, such as m~gn~ m or ~ , from the biodegradable PCR reagents improves their resi~t~n~e to biodegradation during st~r~. Storage of nucleoside 30 tripho~hales and single-stranded nucleic acid (e.g., plilll~l~) without m~..P~;~.n~ is generally observed to improve shelf life, probably ~..~ many of the en~ymes which might degrade them and which often are present as trace co--l~-.-;n~nt~ in reagents and on sllrf~es require ma~ s;.. for activity. The COI c~ tion depPndence of m~r,.e ,;---n activation of nucleic acid l~olyllh, ases is such that low levels of m~n~Sium 35 leakage into a m"~lul~ of the otha PCR reagents should result in n~ligible side reaction, whe,~as side reaction depP,nd~n~e on nllclP~os;de triphosphate, primer, and cnL~Ie concentrations is apl"u~ t~,ly p~u~o~lional to reagent conce~ ion.

WO 91/12342 PCr/US91/010?~'' 2075050 lO
The wax P....hQ~l;...~ nl of the second aspect of the invention is a great impro~ h,nl over the mineral oil l;ul~ lly used to minimi7~ solvent evaporation during PCR, b~au~e wax, unlike oil, does not cling to the pipet used to withdraw PCR
pi~lucl after ~mrlifie~ n and, Ih~er~ , does not c(?~ -..;n~te post-PCR ~et.o~tion 5 re~ctil?n~ According to the cur.rent art of PCR, mineral oil often is removed by time-cou~u~ g extraction with toxic oroth~,w~ hazardous organic solvents. Rep! ~ce-m.o.nt of mineral oil with wax has another advantage quite apart from the function of wax as a vapor barrier and as a means of minimi7ing side re~ nc; the solid seal formed over the ,~acl~n "fi~lul~ after amplification reduces the lilrelih~ of conl~ ;n~l;ng the 10 working envil~."~nt with PCR l"~lucl, thereby l~ g the chance of back-conl---..u~lh~g~ubse~lu~"tl~e1;on~ Highly ~UI~ andpreciselarge-scale nun~r~c~ of wax pellets which are rapidly and accu,~ly ~ pe n~ to reaction tubesduring man..r~ or by the user also i.lwcases PCR conve,~nce and reliability.
The second and third aspects of the invention are impro~ .l,b over the simple 15 layering c~--hQ-l;---f,~l of the fi~t aspect in that they ç~ r the need for manual deAt~ and close ?~tçntion in setting up a co...~ te r~lion Illi~lUA . They also greatly extend the shelf life of complete reagent formulations, so that those fo~mll~tion~
can be bulk n-n ~r~c~ d and stored If the heating of the first Pmrlifir~tion cycle is needed to co...hi~lc all of the PCR l~g_,lb into an active ~ ul., one can ...~ r~ e 20 a ready-to-use reaction tube which C4.~ all reagents and l~u"~es only the addition of test sample and he~ting The c~ )os:~;ons of the fourth aspect of the invention improve wax function in several ways. S~ r; ~ in the wax or grease or underlying aqueous layer reduce the depth of the water-wax ...~ ni~s, thereby reducirlg the mass of wax or grease needed 25 to cover completely the ~ C~"~ layer. A h~r~uphilic co~pos;l;on or coating for the inner surface of the leaclion tube and the inrlllci~n of s~ r~ l in the wax or grease reduce the depth of the wax-air ",. ~ "c, thereby l~Ju~ g the minim~l l~uil~d mass of wax or g~ease. A plastic mesh in the wax or grease reduces the depth of both m-oniC~i and thereby the minim~l ~ui~d mass of wax or grease. This mesh also 30 l~luces the likelihood that a IlliC~)IJi~l used to wi~ w PCR product after ~mplifi- ~tir~n will be clogged with wax or grease and tends to prevent the spurting of PCR product when the wax or grease layer is ~nell~ted. Plastic particles suspended in the wax or grease also reduce the ove.la~. mass, but their major benefit lies ini~lpa~ g a slighdy crumbly texture to the wax or grease, also re~ducing the clogging of 35 pipet tips and the spurting of PCR product.
There are several advantages to n~;--;..-;~.;--g the mass of an oil, grease, or wax layer over the aqueous layer in which PCR amplification occurs. The speed with which '~ gl/12342 PCI/US91/01039 the contents of an i....~.l;r.. -~;on tube a~lua~ t~xl temperatures in a the~nal cycler varies inversely with the total mass of the tube and col.t. ..l~ Absent the present inve,~Lion, the hy~llu~hobic o~e~ often has a mass approaching that of the aqueous layer. M~i,nul,~ cycling speed is highly de;,ilablc in many PCR applications, 5 es~i~lly for clinical ~liagnostirs~ and is ~lull~t~,d by ...;n; ..;,;.-g the overlayer mass.
Thicker layers of wax often resist penetration of pipet tips to wi~ aw PCR product, providing a tight seal around the tip which hlcl~ases the likelihood that PCR product will spurt from the amplificr~ ion tube. Thicker layers hl~l~ the likelihood of plugging a pipet tip with g~ase or wax.
Ease of pipet ~n~a~ on of a wax or grease layer, such that spur~ing of PCR
product is avoided, is highly ben~r~ci~l b~-~use such spurting can ~,~ n~ , product-cc~ ;--;--g aerosols which can c~ -;n~ the labc,latcl~ em~ nt, ~ n~ , and reagents with PCR product. ne~ ~ of the ~A~ e sensitivity of PCR, even trace cnfi~ ;OI- can cause false-positive results in later ~nlrlifir~tion~
Still another, major advantage of the invention is that the invention f~rilit~tes any manner of n~lific~tiûn of PCR ~I.~ ,;n reagents are added late in or at the end of the amplifi~til~n to change the nature of the PCR process, f:-~ilit~t~ the ~aectiQn of amplified nucleic acid, or help to prevent back~o~ -..;n~t;on of subsequ~nt ~n~rlifir~tion~ Such mndifi~ti~n~ include the following: PCR ~Jluducl cleavage, 20 as~ 1- ;c PCR, nested primin~, multiplex PCR,PCR p ~ ucl PhOtO~lC~ ;1;7~t;-~n, PCR product l~beling, and the analysis of PCR product. Such reagents include thefollowing: m~ified p~in~rs~ internal primers, primers dil~t~ at new targets, metal ions, chPl~tors~ new e,l~y,l~s, -~tliti~n~l nucleic acid polym~.~.se, m~ifiPd or lln nuclcosi~le l.;l.ho,~h~s pho~ ag~ 1s~ agents which bind spe~ific~lly to amplified 25 nucleic acid, nucleic acid probes, agents which capture, ~ le or ~g~ l;n~le ~rlified or single~ ded nucleic acid, and agents which help to ~ ale analytical signals l~ ng on the l,lc~nce of ~ l;r,r~ nucleic acid. In every c~, the presentinvention permits such late addition to occur with l~luced concern that opening the PCR reaction tube might ill~duce ~rnplifi~ nucleic acid into the labo 30 en~ilu~ ."
To l,lu~ c und~ n~ing of the invention, ~.-.finitionc for the following terms are provided.
C;11m C~J~ n~ refers to a ~ubs~S~nce cc~ n;ng m~ es~ in a fo~m such that divalent m~..r ~ (Mg+2) is l~leased into any aqueous solvent of pH ~9 35 heated in contact with the s~lbst~nce to a ~ll~lature l:h~n about 50 C and lOO C for an interval of a~p~o,.;...~lt ly 0.5 to 5 ,,,;,,~ s WO 91/12342 PCr/US91/010 2 0 7 5 0 5 0 "PCR reaction tube" refers to any conlA;nf.~ s~lit~lc for hol~ing PCR reagents and test sample during a PCR ~ ;r~ ;f)n In some con~ , the term also comprises the c4nl. nl~ of the conlai"~. The ~Pfiming fealulcs of a co~ .,. suitable for holding PCR reagents and test sample during a PCR ~n~rlir~ ~;on are that the conl~ f,l iS made 5 of a m~t~i~l which does not inhibit PCR, that can will~ m~.at~.,s in the range of about 20 C to lOO C while ,~-;.-ing su~stqntiqlly the same size and shape, and that can, ~og~ with any liquid contents, be capable of cr~mp~ ng 40 C le~ alulc chq~ ges in the 50-lOO C range in an interval of not more than about four ~ ulesPreferably, the c~ .inf" also will have a tightly fitting lid, which blocks water vapor or 10 liquid escape from the c4ul~;n~ as well as co ~~ ;on of the reaction with potentially nucleic acid-co~ inil-~ aerosols and dust from the lr~ / envi,un,,lenL PCR
tubes c~....-..,-~ly are molded f~m polyl,lop~lene, have sizes ~pl~liate to contain 20-200 ~1 reaction ~s, and have a tightdy fitting cap. PCR reaction tubes also c~.. only have shapes which fit tighdy in dhe wells machined in dle metal heating blocks used to control lea.;~ .n~ in most cJ.. ~.cially available dhermal cyclers. PCR reaction tubes most co..... only have dle size and shape of mi.;,~ce..l,iruge tubes widh 500 600 ~1 capacity; the bottom half is conical while the top half is cylinAri( ~l "Thelmal cycler" refers to an al l~!.. h ~ device for controlling dhe PCR reaction 20 t lll~ at~ widlin dle limits ,~u", d for primer ~nn.oqling, primer eytension~ and ~l~xlu~;l d~n~ula~ion; these limits n~rm~qlly are about 40 C and about lOO C. The cycler cllqnge~ the reaction t~ .~y~ in a l~ g m ~ ner, spçndin~ intervals of about 0.2 to 10 ",;"~t ~s at each t4~lul~, and l~ ;ng up to ~veral ~ ..J~,S to move from one ~ f,~ to the next. (~ nnly dheImal cycler t~ alul~ is under plu~.~.. ql~e mi~,ol"ocesso, cont~l, so dhat the user ~ ;es in advance dhe number of cycles, the ~um~. of dirr~ .,t.~ n te---l~-~ s in each cycle, dhe value of each h--nl~e- ~ " the in~ - interval at each h .. -~ , and often the transit time ~l~n ~Ill~lalul~s. Thermal cyclers transfer heat into and out of PCR reaction tubes by con~ ;,-g tubes with circulqting tl.. ,.1-~O~ tf~d air, circulqting 11,~.. o~ ~ liquid, a 30 ~ d metal block, or l;~ l;on from an il-rla.~d or ll~icl~wà~e source.
"Plastic" refers to a polymer c~ ;.-;..g carbon and some comhinqtion of hydrogen, oxygen, nitrogen, flyc rine or much more rarely, other elPm-Pnt~ (such as sulfur), whcl~;n the polymer is fab. ;ral~l into a form (e.g., pellet, thread, sheet, rod, mesh, bead, or tube), which is water-in~luhl~P and substqntiqlly water-i~l.yn_lllleable.
35 For the yulyose of the present i..~el~ , useful plastics include polyethylene, polyyroy~lene~ polyll,~ yl~nt~lle, polyester, nylon, fluc,rvca,Lons, flu~in~q-hydlvcdl~ons, polymethylmPth~q~ylate, and poly~ly,~"e.

wosl/l2342 ~''~ 2 ~ 7 5 ~) 5 a ' ~pcr/us9~ o39 "Plasma ~ " ~d "co~oDadi~ are proeesses for~ g s~ r;.-~.5 lly plas~c s~urfaces, by su~jec~g dlem~ m a ~d ~ ~ of oen at s~u~
~I"~ r- ;G ~ , to a h~ghly ~vc ~crc of Cl~hOllS and att~mic or m~-l~ll.ar ions, ~.,~d by c~ ~on ~thc gas. l~ J;~ on tbe gases 5 p~esent in the ~ ~, Atr~ .. Y~ ~ of the ~urf.,ce may ~esul~
For the ~ ~. of the pIescnt inv~, lhc~mo~rc-wi~ e oxygen or water vapor, so that an ~ A plas~c so~ k~ ~i~y 4 ;~ l and gains ~d hy.hu~.hili~f9.
'liquid hs~ling system" r~ ~ an ~nmalcd dc~ice fa~ ~y and ~ dd;~_~gliq~ ~LS~e~t~io,d~c~angcof 1~ Lco ~y~
some c~. . ~hi~ n of liqu~id 1~.~ ~s, tub~gj deliveIy ~s a~ lc s, h~ nrablc heads or r - ~ ;66~ ~ forp~iomng-d~ dps ~n three c ~ ne, andcon~ol of ~ne, v~. ~ , andpo~ n of dcl;~
"Oil" rcfer~e ~D a w~er-imm~ble , ~q~d at ~ s below about 40 C, which ha~ a lo~densitt,r d~ .wa~r. nluinc~s-l odl", also known as liqmd ~hulalu~l and ~ - H~.~ C~, iS a-col~less, ~*.~lly clea~ , of ~igh-~!.e lllg- weight L~OC ~ ty n~ gh~. widcly avil~ble co- .. ~;&lly and co~ ly use~ as a v~~ ~ o~r ~CR reac~ol~
"Greasc" re~ers eo an ~gP'~- ~stance, sdid ar s~m-solid but very soft at 20 t ~ s below about40 C, ~chmelt~em~be4~80 Cranget~fûrm a liql~id which h~ a low~ density than wa~. A typi~al-~ is white pe~~ lm (e.g., Vaseline~ PetToleum ~elly), a .. .~ , of-l~h-n~ular-weight h~ . L~ c '~Vax" refers to an arganic s~ts~, solid ~ ~L harder t~ greases a~
,s below about 40-C, w~clt melts at some~h~t higheir t ~ ,S to foqm 25 a liquid which has a lower den~ty ~ wa~ r. Waxeg tend ~ a~here to solid (e.g., plastic) smfaccs more wcakly tha~ ~s a~d o~ do. TypicaE p~e co..~ ls which are usc~ul waxcs ~ c ~ c (~, ~~bcosane (C2~sg), cetyl polmito-te (C32H~j402), andpenta~ l t h:~c;~ ~t~ (Cg311lgoo8)- Typical useful wax ~s include p~affin, Parap~t (tr~em~rk of Sherwood Medical), Ultraflex 30 (trademark of Petrolite Corporation), and BeSquare 175 (trademark of Petrolite Cc.llJo,alion). Waxes can bc ~by mDang pElIe or mixed waxes widl one Lc. or with greases or oils in any ratios which pre~erve the relative hardness and ~lUCe~l sti( ~n~s cl~ ~ ;C of a ~
' PCR reagcnt" refers to any of the ~llowing ~;-o-hwhich is nP~e~ for 35 PCR ~q- .~ ';C ~;n~ n-lrl~e ~ph~hate (at 1P~ are t~P~-l for eyorr~le~
dATP, dlTP, dCTP, and d~TPff~ oly~is uscd), ~n~ clp~l;de primer (n-~rrn~lly at least twO are nffll~l ~ng by 9 ~ COmpl~ r to the two ...

WO 91/12342 PCr/US91/010~
20750~0 14 ends of the target s~ ce to be a~rlifie~)~ a ma~..c~il.... co...~ d (m)~n~lly MgCl2), and a DNA poly~lase (n()rm~lly Th~.nn~ z~ ,c C~41 polymerase I).
PCR reagents may include nllcleos;~le ~ hosph~le analogues, such as dITP and 7-deaza-dGTP.
"Test sample" is any liquid yl~ion (solution or ~ ~yenc;~n) which might contain nucleic acid ~,~t~d by primers i~ ded among the PCR Tc~entc~ Wh~ l that nucleic acid is in a ch~-.-;r~l and physical state suitable for PCR, ~nplifiration.
A "subset" of PCR .cag~.lls is any co~..h;n~;r.n of the above reagents which lacks at least one e-~cpnti~l reagent and ul~.~fc~l~, will not sustain PCR ~nlrlifir~tion.
"('omrle .~- n~ subsets" of PCR reagents are subsets which, when combined, CO~ Jht~ the above list and ILel~,ful~ in combination sustain PCR amrlifir~ti~n in the yl~nce of the target nucleic acid s~u~ . Complcll~a~ reagent subsets are said to "cc~ yl~ " one'another.
"Activity" in PCR refers to the ability of a subset of PCR reagents to sustain 15 ~n~rl;r~al;on of a specific target when c~-h;n~d in a PCR l~ion tube with a buffer, the co ~1 ~"~ reagent subsets, and a test sample conl~inin~ that target and subj~l~d to thermal cycling under cc-n~litions known to give qnlrlifi- ati~n when all PCR re~ntC, buffer, and test sample are mixed imm~i~t~ly before cycling. "Full activity" implies that the quantity of specific PCR product ayyl~ t~s the ~..~;"."-.
20 amount ever seen under the particular amplification con-litiQn~
"Specificity" in PCR amplification refers to the ~_, el_~n of a single, "~peçific," PCR yl~lucl with the size and s4uence predicted from the s~uences of the primers and the g~ ~-c,...;c or trqn~ibe~ region of nucleic acid to which the primers were ~e~i~ to anneal in a base-complc~.l~y manner~ "No ~ ; I ic" PCR product has a 25 size or se~ nce dirr~ from such prediction~ PCR "target" is that ~nr~nir or tra~ibe~ region of nucleic acid, the ends of which are base-comple-~ t~ y (with proper o, ;e~t~l jon) to a pair of primers inrl~lded in a c~mplet~ set of PCR req~e-ntc.
"Proper ~liel t~ion" is for the two primers to anneal to opyo~i~ strands of double-stranded target with their 3' ends pointing toward one another, n~rmqlly with an30 intervening region in the ayyr.~ ; size range of 50-10,000 nucleotides~ Such primers are said to "target" the gt-n- mir or tr.qn~ibed sequence to the ends of wh*h they are base-comple.~ ~,nl~.y.
"Layering" of PCR reagents or test sample or solvent refers to the process of delivering liquid fonnlllqtions of dirr~"~inl reagent subsets or test sample or solvent to a 35 PCR ,~,aclion tube in a way which ~ es their mixing without int~.~osition of an ;--.l-e ~--e~ble barrier (e.g, of wax or grease) bCL~ them.

'O 91/12342 PCI/US91/01039 "Vapor barrier" refers to a layer of oil, grease, or wax on top of the aqueous CcJ~ t of a PCR reaction which covers a large enough fraction of the exposed nql]~POll~;i COl~ IlllCnl surface to reduce substantially evaporation of water from that C~InPZIL~ during thermal cycling. Vapor barriercoverage is "c~mp!ete" if, for an5 ;~quews cc,~ .~nt of a~~ ;..L~t,1y 20-200 ~11, 30 cycles of PCR amplification distill no more than about 2 mg of water to the walls and cap of a PCR reaction tube above the level of the vapor barrier.
"Liquid barrier" refers to a layer of g~ease or wax, which, at t~ lu.~,s up to at least about 40 C and not more than about 90 C, sllfflçes to block mixing of aqueous 10 cc,~ ,a,t~nls on either side of the layer for an interval of at least al)p~ ely 15 Jt~S Liquid barrier coverage is "complete" if this 15 minute critPrion is met.
"Thermal cycling" in PCR refers to the process of ~ h- ~.AI;c~1ly and repetitively çh~n~ng the temperature of a PCR led~lion l~ lUl~ in the a~ .;...AI~
t,~lalul~, range of 40-lOO C to effect alt~ a~illg delulu ation of doublt-stranded 15 DNA and primer ar ne~ling to single-stranded DNA followed by primer eYtPn~ion.
"S!-- r~ " is a subst n ~e which reduces the intPrf~~iA1 tension ~t~.~n water or aqueous solutior-c and }.~ phobic solids or liquids like polyolefin plastics, oils, greases, and waxes. Surf~~t~nts are co...pose~ c~lly of covalently joined hydl~philic and hydl~phob ~ moieties. "N- nil~ni~ s~ rtqnt~i" contain no positively or 20 negatively cl-dl~d rnoieties. Typical nnnil-ni~ r; ~ include the following fiqmiliP,s of sllu~tulill ho-~olo~ues. "Span" (t~qAPmq* of Atlas Chemical Tn~ tries fatty acid rnono-, di-, or lliestcl~ of sollJi~); "Tween" (~ .1. of Adas C~hemic~l Tr.~lv-~ ;es. ~olyu~ ylene edhers of fatty acid esters of sollJilan); "Bri;" (tr~ lPmqrk of Adas Chemical Tn.l..-l. ;~; ~olyuA~_Ih~ e edlers of fatty alcohols); "Myr;"
25 (1~ A~ ..lr.l~ of Adas C'hPmicql Tr.(1..~1. ;es ~O1~A~ lene esters of fatty acids); and "Triton" (I.~d~- .. -.1. of Rohrn and Haas ~ornpqny: alkylaryl polyo~ llyl ethers).
Specific nonionir s~ ct-nt~: which are ~l~f~ d for the present invention includeTween 85 (polyoxyethyl SOlbil~l I ~le~t~,) and Tween 65 (PO1YUA~ Y1 s~,llJit~
lliS~ualc). These two CO~ lcially available sll~Gt~nt~ are just exarnples of a 30 ~lluclulal class known as "polyo,-~elllyl SOllJilan triacylates," wh~lcin polyu,~yc~lyl chains of various lengths and dlree fatty acid residues of various sizes and structures are covalendy qnq~hed to one SCIlbil~ rnoiety.
For the pwl~ose of dhe present invention, a "hydlul)hilic" surface is one which,when on dhe inner wall of a tube, shows a concave-upward mrniscus when water partly 35 fills the tube.
ll fatty acylate salt" refers to a co,n~ilions co..l;~ g a 1:2 mol.,r ratio of Mg+2 to dhe conjugate base of a fatty acid. Rc~l~scll~ti~e fatty acids include butyric (C4HgO2), caprvic (C6Hl202), caprylic (CgH1602), capric (CloH2002), and lauric (Cl2H2402) acids.
Choosing PCR pnmer se4u~nces, ~ g DNA-co~ ining test samples, PCR rea~ntc, and PCR reaction ~lu,~,s, ~lesigning and running PCR thermal cycles, 5 and analyzing PCR pr~duct ~ ely or qu~lit~tively are well known procedures in PCR ar~ A ~,f~l~d mode fvr carT~nng out the first aspect of the invention is to cc~..h;ns in the PCR reaction tube a buffa, poly~ ase~ dNTPs, and ~ in an eo~c sol~lti~ applv~ ely twice as co--r-e-.l. ~1 as is desired in the final reaction ~lUlG and to layer on top of this "pre-mix," in any order, a stock solution of MgC12, 10 a teGst sample believed to contain target DNA se~uence~ and s~ffl~e-nt mineral oil to foIm an effective vapor barrier, such that the c~...l.;nr~ volume of MgCl2 and test sample apprv~ ,s that of pre-mix, and the final MgCI2 conr~ntration is apl)lvAilllately optimal fvr ~q~l;r~cation of the specific target s~ -ce defined by the primers. Altho~lgh the prG-mix is ~lb~l-nl;~lly m~..~s~ free, the pre-mix may 15 covntain m~g~ m at a conc~ ;on less than abvut 1~4 M without ~ h~ing PCR
~mrlification in advance of adding the much larger amount of ...~.es;~ . initially sc~Eat4d frvm the pre-mix. A plefell~d vrder of layering is to 1n~1~0SG the testsample ~l~. ~n the pre-mix and the MgCI2, adding the mineral oil last, as this vrder mpXimi7es the segregation of the complc~n~ reagent subsets. Layering preferably 20 is ~lÇvl~d with all cn.~ on~nl~ at~vom t~ ,-atl..G, delivering the com~)oll~ tO
the walls of the reaction tube slowly, so that minim~l mixing of the dirr.,lc;n~CQ~q~Qn~-n~ occurs during ~lition~ Preferably, reaction tubes are made up in this way within about 30 minutes of starting anq~l;rlc~ n (the shvrter the time the better)~ Also preferahly, the first step in thelmal cycling will be the fastest poscihl~ heating of the PCR reaction tube f~vm room te-.q~ ; to 90 100-C, to ensure rapid and c~mplet,o,convective mixing. The stability of reagent layers before heating can be incl~,ased by incol~,vl~ g in the lowest layer a cl~e~;c~lly inert, nnni-niC, ~P.n~ifier such as sucrvse at a corcenl~ ~on ~I~.~n about 1% and 20% by weight.
The rep~ .. -t in the PCR reaction tube of mineral oil by grease or preferably 30 wax leads to the second aspect of the inv~,n~n,which is a pl~,Ç~ ,d mode of effecting the first aspect of the invention. Once a layer of wax or grease seals the top of an aqueous solution of a subset of PCR reagents ("pre-mix"), an aqueous solution of the compl,~.~ reagent subset (pl~f~ ly in~lu~ing a m~e~ m salt) can be added on top of the wax or grease without any COI ~ ~- that poor manual control will result in 35 reagent mixing before thennal cycling begins. Test sample can be added before or after the wax or grease barrier is formed. Adding test sample aft~,l w~d usually is more convenient, if only bec~n~e large batches of reaction tu-hes co"l;~;n.ng wax- or grease-'O 91/12342 PCI/US91/01039 17 20750~0 covered pre-mix can be made up ç~ in advance and stored for periods of days to mc-nth~, preferably at 0-5 C, so that test c ~ ..ples are added only as PCR reactions are nee~ed In this ..u~ r~ g mode~ one preferably adds on top of the wax or grease barrier the reagent(s) micsing from the ~qneovs laya beneath the b. rrier after the b~ier 5 has been formed and before st~rage, so that the user needs only to add test sample ;.. ~A:~tr~ly before thermal cycling. If the manufactured tubes contain everything except test ~q~npl~, the wax or grease barrier must be durable enough to prevent reagent L. across the barrier on the storage time scale, as some wax or grease layers may be strong enough to leak n.o.gligibly on the time scale of an hour or less but show 10 ~ir~;r;r~ ~1 leakage on the time sc.le of a day ormore. One way to test leakage across the barrier is to include in the upper aqueous layer a water sol~lble, wax-in~ ~ dye such as lh~ .k~ .-ol blue at a cQnf~nl~Lion of 0.01% to 0.1%".1,c L ;--g visually for signs of dye .l.~v~ cnl into the lower aqueous layer.
A ~f~ ,d mode of creating the wax or g~ase barrier is to add to the reaction 15 tube a so!vtic!n of the reagent subset which is to lie b~n~qth the barrier and a mass of wax or grease sllffir;~-nt to form a vapor ba~ier and then to incllbate the tube, preferably closed, for ~ e-ll time at a s~ c:ent t~,.n~l~lul~, for the wax or grease to melt and form a ho..~ ous liquid layer above the -q-q~leoll~ reagent sol~ltic~n The tube is then ~ lod to room tem~ab~re for at least enough time for the wax or grease 20 to solidify. The; ~u~';~n t ---~ u-.~ must be above the wax or grease melting point but preferably will not be more than 10-C above the melting point and in no case above about 90 C, so that the possibility of thermal inactivation of pol~ cko! ;~le polymerase, if present in the pre-mix solvtirn, is ~ d Narmally a melting time be~
about 30 ~-4nrls and about five ~ ,t~ s should suffice, and a cooling time of at least 25 about five ll.;llut~s is plefe.l~d for the barrier to harden completely. Rapid cooling, for eY~mple in an ice bath, appears to be unde~ beca.lse the wax or grease is less likelytocoverthel,nd~ ringP~Iu~o~ ~nt.
The mass of wax or grease preferably is l..il~;...i~l. using just enough to ensure complete coverage of the aqueous con~nt~ of the reaction tube after melting of wax or 30 gre~ during dhermal cycling has resulted in mixing of dhe aqueous layers above and below dhe barrier layer. Such coverage can be a~sesse~ by ..~ g how much water iS ~ tillf~ onto the walls and into the cap of the reaction tube during a PCR
amplifir~tion For example, after an~l~!;r~ ;ol~ the tube can be weighed (preferably on an analytical balance accul~tt; to 0.1 mg or less), the water depo~ilcd above thé vapor 35 barrier can be removed by gende swabbing with a cotton-tipped stick, and the tube is then reweighed to ~ f the water removed. For ~nd~.l PCR reaction volumes (20-200 ~1), complete coverage results in ~i~till~tiQn of less than about 2 mg of water to WO 91/12342 PCI/US91/01 o~r the tube ~u r~ces above the vapor bar,rier. In general, the minimql mass of w. x or grease l~uil~ for complete coverage (,.~ l vapor b~rier effectiveness) will v~y directly with the tot. l aqueous volume because the conical crvss-section of typical PCR
reaction tubes causes the surface area at the air-water ;~t~.. r;~e to in.;l~asR . s the 5 .~ ue~s volume incl~ases. There are at least two reasons to minimi7e the mass of wax or grease: (1) the time l~uu~d for thermal çqllili~tir~n during c,vcling varies directly with total mass of the reaction tube and its co~tenls~ and (2) wax or grease in excess of the ...;-.;...--... mass needed to senre as an effective vapor barrier cQmI~lirqtes the recovery of PCR p~lucl after~thermal cycling, tending to clog the tip of the ll i.;lo~,ipe~
10 used to willl~aw PCR ~ ducl from the tube or (in the case of wax) creating a Ally tough bar,rier l~";~ ..-g c~n~sicl~ ble ~ ul~; to effect ll i.,lv~ipel tip pc.l.,halion. Ncrm~lly the wax or grease barrier is pe~--,h~tcd for sample withdrawal simply by applying light manual p~ss~ , to the top of the barrier (usually at the center, where it is ll~;nl~e~) by an o~dinary air~ or positive-dis~l~c~
15 lllLlvp;~l or "sampler." IIv.._~_" a wax baIrier also can be bl-_aclled by freezing and thawing a wax-covered ~queous layer. The eYp~n~ n of the ice Laclul~s the wax sothat pipets easily ~n~,h .~te it; a short cçntrifilge spin in an angle rotor helps to assure wax fr~ ;on Wax is ~l.,f~"l~ over grease ~ e wax is much less likely to clog or coat 20 ll~l~i~l tips. Being tougher than grease, wax can effectively segregate PCR reagent subsets in a thinner layer, and, Ill~,erol~;, with less mass. Many wax collll,osiliol s are effective fvr the pul~lose~ of the present invention, inf~l~..ling the following: ~rm (55~1-C melting range), ~ c~ ~e. p~pl~ct Ult-~fleY o~n ~4C~C, cetyl pqlmit~tç
penta~ l.lilvl ~t~ hen~l~ and "BeSquare 175" Wax. Mixtures of these waxes in 25 various plvlJvllions~ ~lul~,s of these waxes with grease such as white petrvlatum, and l~lul~,s of these waxes with mineral oil also may be advantageous. ~lthough individual wax pl~alalions may prvve lm~lit~e7 either b~ ~ they contain sub~l~nr,es inhihit~ry to PCR or because, coming frvm biological sources, they may contain C~ t~ ting DNA capable of giving false positive PCR l~vnses, in general 30 almost any wax melting ~t~. ~" about 40 C and about 80 C might serve as both a barrier layer fvr se~ ~a~ih~g PCR l~c-~ befvre thermal cyding and as a vapor barrier for ...;~ ;ng water e~d~la~ion during thermal cycling. Waxes, greases, or oils p~ ,d frvm petroleum are plef~,l~l over materials derived directly from ~nim~lc or plants to ...;..;...i~ the chance of co ~l~ ";~tion with nucleic acid, phosph~t~es, 35 nucle~es, or ~lotcases. They must have ~e~ ;es less than that of the aqueous solutions used in PCR if they are to serve as a vapor barrier during amplification Heavier-than-water waxes and greases (e.g., cont~ g silicon, phosphorus, sulfur, or ''O 91/12342 PC~r/US91/01039 19 ~0750~50 halogen atoms) also would save the function of s~,alaling PCR reagent subsets until the first arnplific~tion cycle. Although unlikely to w~ as a vapor barrier after he~ting, they might ~.~,r.ci~lly sink to the bottom of a reaction tube when a vapor barrier is not neeAeA and in doing so might l,lull,~lc mixing of previously sc~ aqueous 5 re~gent~ However, layering a heavia-than-wata molten grease or wax over an queou~ co.ll~)&l.lh,nl l~Uil~,s cQ-~ 1e eA~lise.
The siln~)le;,l way to test the s~li~hility of a waA for the present invention is to (1) colll~ine in a 500 ~1 poly~ yl~ l~e..n. ;r.l~e tube appl~ ely 15 mg of the wax and a~plv~ t~ 50 ~1 of an aqueous s olutinn c~nt-;ning all PCR reagents except a ma~.~ .. cc.. ~ n~, (2) cap the tube and ;.. ~--~ the tube in a water bath at a temperature ~ .c~.l;.~g the wax melting point for a s~lfficient interval (a~ rly one minute) for the wax to melt completely; (3) remove the tube from the water bath to cool to room ti,~ alul~i, (4) add on top of the wax ~ t~,ly 50 ~1 of a ll~lulc of MgCl2 and a test sample u~fil~in;t~g DNA targeted by the primers included in the 15 PCR rea~ .nt~ (5) ~Irullll a PCR ~ !;rlcation using thermal cycle conditions appl~liale for the particularco~ in~;on of primers and target DNA; and (6) analyze the post-PCR l~~ti~n ~lul~ for the ~ ~nce of PCR product of the predicted lengthco~ ;n;ne a predicted intervening (non-primer) se~lu~,nce by m~th~ well known inthe field of m~le~ sr biology. The c~ l-~nl~tions of all PCR reagents and of target 20 DNA should be within ranges already ~ e-~- od to give errecli~,e smrlifirstif~n when mineral oil, usually in the mass range to 40 to 80 mg, is used instead of wax.
During this testing procedul~" several adjunct analytical ploce~lwes help to optimi~ the results. Visual examir~sti~n of the wax layer before adding MgC12 and DNA and after thermal cycling helps to verify that the mass of wax is large enough to 25 serve the liquid- and vapor-balTier funr-ti~ ns If the mass of wax used is in~llfficient to cover the a~ueuus layer cornpletely, the mass should be illcl~ased until coverage of the ~ueol~s layer is complete. Gravimetric ~aswe~nt of the water ~istilw past the wax into the upper regions of the reaction tube (by weighing the tube before and after using an absoll~nt swab to remove the water) also helps to evaluate vapor barrier 30 effectiveness. Di~till-stion of more than about 2 mg of water i~-~ic~s that increasing the mass of wax may help. Probing the h~d,less and toughness of wax layer with amiclu~i~l tip may also in~;~ste whelh~ the wax ~lep~u~ion is useful for PCR;
preferably the sampler tip will penetrate the wax without clogging or causing wax to stick to its sides, and ~neh~tion will require little force and be easy to control. Certain 35 anionic dyes, such as l~l~n~l)hf .~ol blue in the conre~ tion range of about 0.01% to 1% can be in- lllrl~1 in the aqueous layer above the wax or grease wilhoul int~lr~ling with PCR. Visual in ;pection of how well the dye has mixed in the aqueous layer WO 91/12342 PCI/US91/0103'' 207505~ 20 below the wax or grease after the first cycle is effective for testing that the wax or grease does not impede mixing.
These tests enable one skilled in the arts of PCR and ml~leculqr biology to choose waxes, wax l~lulcs, and u~luu~,s of waxes with oils and greases which serve 5 optimqlly for se~e~aling PCR l~ag~r.b and for bla~'Ying solvent evaporation, and to find the optimal mass of wax for a p~u~;ul~ co...hil-G~;~ n of aqueous volumes above and below the wax barrier. If, in addition, a wax formlllqtir~n and mass are needed to confer long-term storage stability to a wax-covered PCR reagent subset, with or will~ul the long-term ~ nce of the comph~lku ~ subset above the wax layer, one 10 need only pelr~ these tests after dirf~ nl storage intervals ~h.~n pl~A~ 1;on and themlal cycliflg,'preferably adding any miCcing reaction c4..q~ nt~ (in-~1...1il-g target DNA) on top of the wax layer just before thermal cycling.
F.spe~qlly ~,~f~ d for t'ne second aspect of the present invention is a solutionin the wax of a surfactant, pl~,f~,.~l~ noni~nic in the conren~ ;on range of 15 a~ ;...Ately 0.1% to 1% by weight, ~ d by melting the wax, adding the s~rf5q~tAnt, stirring the l~ lUl~ for a period of at least about one minute and then allowing the matter u~lu~ to stand lln-lictllrbed for at least about 10 ...~ tes before observing ~helllcl any ~,-.. r~rlAi~ has formed a sepdl~ liquid phase, usually denser than the wax. Phase separation shows that t'ne n~minAl surfactant concen~Tation 20 c-.ce~ d the solu'c ility of s~ r~ -- in the wax, su~estin~ that a lower nQminAl conre~ 1 ;on should be use~ ~ ~litinn of surfactant generally lowers the mass of wax needed to form a complete liquid barrier and a Cl~ kte vapor ba~ier, shown by titrating the ...;..;...~ .. mass of wax needed to obtain complete coverage of an und~,~lying aqueous layer, as d~sc- ;h~ above. ~fu.l~d surfactants are ones which are 25 not very water soluble, con~ .;..g l~ ely small h~ul)hilic moieties and relatively large hy~llu~ho~ic groups. Sp~ ;rlrAlly p~f.,.l~d cou,~ ially available ~... ra ~ nl.~ are Tween 85 and Tween 65. Novel PO1YOA~ }lY1 soll,i~n triacylate sllrfa~tAnt~ with polyoAyelllyl chain length or fatty acid sllu-;lurc ~~ ~ for PCR vapor barrier pe.rO....An~e may improve pe.rJ- ~-~nr,c in various ways. For example, PCR thermal 30 cycling of ~ ,o--c solvent beneath a ~u~r~ant~ nil~g wax layer may result in some extraction of surfactant, possibly ncc4...~ -ie~ by wax, into the aqueous layer.
This event can be analyzed by ~f A~ g the ultraviolet and visible absoll,ance spectra of the aqueous layer, as e AIlacl~d surfactant forms m;~ellAr particles with a Cll~ et~ ~ ;Cti~ light-scal ~.. ;ilg ~11 l~lll. ~lthough eAll~d ~ -- rh~ does not 35 gçnerally i~te.r~ with PCR or post-PCR analysis, it may be ch,~ ~A1 in specific es Sh~.lh-g the length of the PO1YOAYCIII.Y1 chain or increasing the length of the fatty acid from what is used in Tween 65 or Tween 85 should reduce surfactant 21 20750~0 water sol~lbility and extractability during thermal cycling. Use of the second aspect of the invention may be in~ vtd also by inccl~ ;nn into the wax or grease of an oil-soluble dye such as Oil Red O, Oil Blue N, Solvent Green, Fat Red, and Sudan Orange in the con~ alion range of about 10-3% to 10-1%. Such dyes do not in-~r~ ~ with s PCR.
T~ n in the bottom aqueous layer of 0.1-10% of a surfactant, also ,f~al)ly nnni~niC, may serve to iu~ u.e the second aspect of the invention, again by l~Ju~ the mass of wax needed for complete coverage of the aqueous layer.
I~GÇe.,~,d surfactants are relatively water soluble ones, such as Tween 20 and Triton X-10 100, many of which do not appear to il,te,r~; with PCR.
Also esperqlly ~,f~ ,d for the second aspect of the invention is the use ofplastic PCR rGaction tubes which have been m~nllfq.~tllred or treated in such a way as to have a l.~d,uphilic int~nql sllrfMe This c~"bo1;~ n~ serves to reduce the mass of wax or grease needed for liquid barrier and vapor barrier filnctiQn Specific p~cesses for 15 making such tubes include (a) plasma etching or corona discharge in a ch ~ . where the open tubes are e-l ose~ to an o~ g all~o~hc.~" (b) in~bal ;~n of the plastic in a liquid o~;~1;,;..g formlllq~iQn such as Fenton's reagent, (c) inCl~lciQn of a ~... r~ nt in the molten resin from which the tubes are cast, and (d) coating the tubes with a~u~ r~ S!-- r~ coating of n~nqlly h~ophûbic ,~;~,on tubes may be 20 accc,.npliched by the following steps~ olutiQn of 0.1-10% (by weight) of the s... r~ in a non~ueous solvent such as 1-p,upanol, 2-propanol, or 1-butanol, (2)filling of the open tubes with the s-~ rS,~ solution at 20-30 C, (3) inc~l~qtion of the filled tubes at 20-30-C fr~ an intenral of ~t~.~n about 1 and 30 n~ S, (4) drainage of the incubated tubes to remove all bulk surfact, nt sohltinn~ and (5) air drying of the 25 drained tubes at -~t...~ .h- ;~ or ~luced p,~s~, at a~ ;.n~lely 20-60 C.
Not all ~--- r~c~ are soluble in useful no~ ueo~s solvents to conce-.l . ~ions as high as 10%; some care should be used to assure that coating s... r;~ t is completely dissolve~ This coating ~ may be ,.,~ated at least about 10 timeswith the same ~... rA~IA~t sol~ltior,, as it appears not to con~lme a ~ignifi~o-nt faction of 30 ,... r, '~ t of each cQoting. F~,f~ d nonaqueous solvents have boiling points bclween about 60-C and llO C and dissolve ~ r~A-n to a cQnc~ .i.t;on of at least about 1%.
f~"~d ~--- r~ IA-II~ are Tween 65 and Tween 85. Corona discharge and plasma etchn-eth~c for treating plastics are reviewed in the following articles and books:
~offrnAn, 1988, JOmT A1 f A~plied P~"h r Science: ~plied Polymer Synl~)osiu 35 ~:251-265; ~olllbo~ and ~ ffmqn~ 1987, C.R.C. Criti~ql Reviews in Bioco~ )AI;hilit;y 4:1-42; Boenig, 1982, Plasma Science ~d Technolo~y. Cornell Uni~e.~ Press, Ithaca; Oskam, 1984, Plasma E~ ~ of MateriAl~ Noyes WO 91/ 12342 PCI /US91 /010~
~0750~~ 22 Publi~tions, Park Ridge, New Jersey. Fenton's reagent is ~les~ribe~l in the following articles: Walling, 1975, Acconntc Qf ('hPmir~ f ~rch 8:125-131; Graf ~ al., 1984, Journal Qf BioloFi~ h~mi~t~y ~2:362~3624; and Imlay and Linn, 1988, Science ~Q:1302-1309. When the second aspect of the invention is ~.Çul~ed with 5 hy~llûyhilic reaction tubes, a pl~fe~l~d mode of supplying such tubes ready for use is to deliver t,o each tube a pellet of wax ~ e.~1 in mass to cover compl~tely a specifiç~l aqueous reaction volume. Then the user needs only add an aqueous sol~ltion of a subset of PCR reagf~nt~, heat the tube several ~ 5 at a t~,...p~ u~c 5-lO C above the wax melting point, allow the tube to c~ol to room t~ u~ ~ or below, add on top 10 of the newly formed wax layer a ~ co- t ;~ g the complementaTy PCR reagent subset and test sample, and start PCR ~n~lification, ~ ~;1;..g the need to keep the total aqueous volume below the rating for the mass of wax supplied.
The second aspect of the invention is most effective if, during the first cycle of thermal cycling, the reaction tube is heat;d as rapidly as possi~le from an initial t~n~ c of about 0-25 C to a DNA de ~ ;n~ If ~ , above 95 C and below lOO C, pl~r~.~.bly about 98 C, and is held at tha~ high tclll~.alulc for an interval l~l~~n about 1 and 2 ..~ ulf-s This process serves not only to melt the wax or grease and to denz~ the DNA in the test sample, but also to promote vigorous theImal convection Cull~ n~ in the newly co...bi~f~l upper and lower aqueous sollltion~ so that 20 all PCR reagents and the test sample DNA are cc~ let~ mixed; incoml~lete ~lule can reduce ~q~l;r~r ,1;m~ f ffiri~ncy. Other e-~ n~C which help to ensure complete ~ lulc are to include il~Ol~liC salts such as KCl in the upper PCR reagent subset and to exclude them from the lower, complc.lh,.,~ subset, and to include a 1-20%
co~-ce.-ui.l;.~n of a low molecular weight de.,sifjing agent such as sucrose in the upper 25 ~uf~OuS layer. In general, it is useful to render the upper aqueous layer denser than the lower one, preferably ~..rlu~ling from both layers organic polymers or inert proteins such as gelatin, which tend to il.~;l~E viSc~ily and thereby reduce convective turbulence. Still another way to ~ l~le convective mixing of top and bottom aqueous layers after the wax or grease has melted is to tilt the reaction tube at an angle of 30 ~l~n about 10 and 30 degrees from the vertical after the wax or grease has been melted, allowing the barrier layer to solidify at this angle. After cooling, the top of the wax should be ~ f ~ visually to assure that the bottom ~quoou~ layer is completely covered, as tilting tends to thin the ba~rier layer.
The wax or grease layer of the second aspect of the invention can be thinned 35 further by incorporation into it of polymeric particles or of relatively fine plastic mesh, two additives which also can reduce clogging of sampler tips. The mesh must be cut, conveniently with a punch, into disks with a .1; ..~. h~ a~yl~A;n~ y equal to the inner CA 0207~0~0 1997-12-29 diameter of the reaction tube at the top of the lower aqueous layer. As the bottom half of the customary tube is conical, this diameter, on the order of several millimeters, will depend on the volume of the lower aqueous layer. The composition and pore size of the mesh tolerate wide variance. Acceptable plastics include polyethylene, polypropylene, 5 polymethylpentene, polyester, nylon, and various fluorocarbons; the pore size may vary from about 0.001 to about 1 mm. Nylon is somewhat less prer~ d, because nylon can bind nucleic acids, and fluorocarbons are somewhat less plerelled because of greater density and cost. Polypropylene is most pler~ d, because it has the lowest density (most compatible with wax, grease, or oil), is highly hydrophobic, has a thermal tolerance 10 matching that of the most commonly used PCR reaction tube material (also polypropylene), and is likely to be the cheapest. Polyme~ly~e~ e is also especially pl~r~lled because of low density, high hydrophobicity, and high thermal tolerance, but pre-m~mlfactured mesh may not be readily available coll"ll~l~;ially.
A wide variety of mesh compositions and opening sizes is available from Spectrum15 Medical Industries, Los Angeles. The shape, composition, diameter, and porosity of the polymeric particles also admit wide variance. Shapes may be irregular or spherical (the latter usually described as beads). Compositions can include any of the polymers listed above for meshes plus two polymers commonly used to make beads: polymethylmeth-acrylate and poly~lylelle. Diameter can range from about 10-3 mm to about 1 mm. Larger 20 diameters (above 0.1 mm) are pl~r~lled because they are less likely to enter micropipet tips. Nonporous particles are preferred because they offer a much lower surface area to entrap PCR reagents which come in contact with the wax or grease layer. Preferably the particles have a density less than or very close to water, so that they are unlikely to drop from the wax or grease layer through the aqueous layer when the former melts into an oil.
25 Examples of commercially available polymeric particles suitable for the present invention include polypropylene granule, 0.5 mm maximum size, from Goodfellow (Malvern, PA);
chromatographic grade polypropylene and polyethylene spherical particles of diameter between about 0.01 mm and 0.15 mm from Polysciences, Inc. (Warrington, PA); Bio-BeadsTM SM polystyrenedivinylbenzene and acrylic ester adsorbents, 20-50 mesh, from Bio-30 Rad (Richmond, CA); AmberliteTM XAD polystyrene and acrylic ester adsorbents, 20-60 mesh, from Rohm and Haas (Philadelphia, PA); plastic or glass-coated plastic BioSpheresTM, 0.1 to 0.2 mm diameter, from Whatman BioSystems, Inc. (Clifton, NJ);
Rapid CellTM p plastic or glass-coated plastic beads, 0.15-0.21 mm diameter, from ICN
Biomedicals, Inc. (Cleveland, OH); and PolybeadTM poly~ylel1e microspheres and 35 polymethylmethacrylate beads from Polysciences, Inc.

WO 91/12342 PCr/US91/010?-Some of these pol~ ic pallicles, espec i~lly those made of polystyrene or polymethylmeth ~ylate, will swell when co.~ o~d with low- -le~ r-weight solventssuch as water and ~ l~oh~ ,f~ bly, they are swollen at room t~ at~llc in a relatively water-insoluble organic solvent such as a liquid ~lirh~ hydrocarbon (purc 5 or a ~ ul~" such as petroleum ether or ligroin) so that they will not tend to absorb water at the ;n~ ces ~h. ~n the aqueous layers and the grease or wax. Some of them may be supplied as aqueous $hl~ies~ already swollen in water, in which casepl~,f~labl~ they are d~ at~d by stirring atroom te .~ in ~ucces~ ely less polar solvents like et~ q~~ et~ and a liquid sliphqtic hydrocarbon.
Thé c~n~en~ n (in mass percent) of pol~l~.ic particles in the grcase or wax permits c~n~ ~ble v~ri~1 ility and can be OP!;n~;~ for any of several filn~tion~l ~lu~ ~ies of the particle-wax/grease ll~i~lUl~. For example, various mass ratios of particle to wax or grease can be tested to find the one which provides complete vapor barrier ~l~t~ ! ;nn to a fixed volume of the und~lljing aqueous layer with the least mass 15 of o.~ .la~r. On the other hand, various mass ratios can be tested to find the range which imparts ,..-~;n.~ .. or ~ nce to ~n~ n by a mi,lupil~el tip or Ill~illlUIll stability during long-term st~age (for example, ..~ .. resistance to m~..e,;~... pen~ ;on from an upper aqueous layer into a lower aqueous layer se,p~ t~d by a s~ ifi~ mlAl"l., of polymeric pal~icles and wax or grease).
Wax or grease barriers cQnl~;n;~g polymeric particles and esperiq-lly disks of plastic mesh may benefit from soli~1ifirqtiQn with the PCR reaction tubes tilted 10-30 degrees from the vertical, to ~JlUll~UtC mutual .1i~ Pm.-nt of the ba~ier and an aqueous layer above it when the wax or grease in the forrner melts.
There are two major e-..k~ of the third aspect of the invention: (a) 25 aqueous çmul~i~n of a subset of PCR reagents in a wax or grease ba~Tier covering an aqueous s~ tiQn of a difr~ t reagent subset and (b) solution in the barrier of a PCR
reagent subset. The melting of the barrier which ~Gc~ n;Ps th~mal cycling breaksthe em~ ion~ ;ng the a~ueo~s phase cm.l ;ninE PCR l~ S into the underlying q-qlleal~ layer, where thermal convection mixes all PCR reagents together with the test 30 sample, preferably added as an aqueous solntion above the barrier layer before cycling is started. Alternatively, dissolved PCR l~ age.lls in the balTier layer are extracted and mixed into the underlying aqueous layer, as the barrier layer melts and hecomes less viscous and as thermal convection CW1~,- ts are est~hli~he~ in both the underlying ~ q leou~ layer and the barrier layer. For these emhotlimr nt~, it is ~o,spe~iqlly plerc.lGd to 35 .";n;".i~G the rnass of the ba~rier layer by any of the modes ~les~ibe~ above, singly or in c~n~inqtion polymeric particles or mesh or ~ tS~nt in the barrier, hydrophilic inner surface of the reaction tube, or ~ -. r~ nt in the aqueous layer. ~in;...;,;.-g the 'O 91/12342 PC~r/US91/01039 barrier mass, and, ~ efol." thirl~n~ss~ acc~ t S the equilibration of the aqueous . nd barrier layers and favors yall;l;O~ g of any barrier~issolved PCR l~iagellb into the aqueous layer.
Water-in-grease or water-in-wax çmul~i-n~ are made by metho~l5 well known 5 in the field of mqt~riql science, basically by high-speed mixing of apployliate volumes of an &~lucous phase (in this case cQ~ ;..ing a subset of PCR reagents) and a melted grease or wax at a l4111y~1at~ above the grease or wax meldng point, followed byrapid cooling of the h~m~g~ vs emlllc;~ n before it has a chance to yhase-scy~le.
Inccnyul~liol~ of a surfact~nt into either phase or both helps to stabilize the çm~ ion 10 Any of a large array of ~ - r~ , preferably noniQnic7 are effective to this end, provided that they do not inhibit PCR and are used to co-~ce~.l. dtions stqhili~ing water-in-oil rather than oil-in-water em~ n~ The art of forming çmnl~inn~ is reviewed in the following volnmes: Becker, 1966, Fmlll~ions: Theory and Practice. 2nd Edition, 12einhQItl New York; Shçrmsn~ 1968, Fmnl~ Science. Academic Press, New York;
15 and ~ i~nt 1974 (Parts I and II) and 1984 (Part m), Fmlll~ion~ ~ Fm~ ion Technolo~y. Marcel Dekker, New York.
~ lthough pla~,~i~lly any colllbination of PCR l~ ,nls is suitable for incc,ly~ldtion into a water-wax or water-grease em~ n, one reagent, a m~ eci~
co~lyvulld~ is most yl~,f~ d for Ai~luti~n in the wax or grease in the ~hsence of e-mnl~ific~tion. That is bec~. se salts of .. ac.. ~-~i.. ion and the conjugate bases of relatively short-chain fatty acids, such as butyrate, cdpl~ta~t;, caprylate, caprate, and laurate, have the dual pl~,~.~ies of being ac~eFtrhly soluble in oils to the level of 1-S0 mM and of being much more soluble in hot water than in cold. In this ~r,~,l, them~gnes;~ fatty acid salt can be Aissolved in a molten wax or grease, layered on top of 25 an aqueous phase and fairly rapidly cooled (in an interval of about one minute) so that it is trapped in the hardened wax or grease before it is e~lla~,t~ into the warm water, and yet will be efficiently eA~ xl when the multi-layered IlliAlUl~, is heated to 95-98 C for a period of one to two ~...~ hs at the start of thermal cycling. To .~;n;..~ , extraction into the aqueous layer during casting of the barrier, it is ~ ,fcll~d to use a wax or 30 grease with the lowest ylac~ical melting point, preferably in the range of about 40-SO C.
Examples of such waxes include eicQs~nç (m.p. 36-38 C), low-m.~lhng p~rln (m.p.
56-61-C), and ~ AlU,cs of these two waxes or of eicQs3n~ with other, higher-melting, waAes. Oligonucleotide primers and dNlP's also can be l~,nd~,lcd wax-soluble if ll~ulsrull~led into their trialkyl~ .. o~.;. .. (e.g., triethyl~l"l~niull,) salts~ This reaction 35 can be ~lÇulll~d by passing the nucleo~ide, colr....ol-ly available as its sodium salt, down a chlulllat/>~)hic column c~nl~;n;ng a cation eYch~nge resin, such as Dowex50, which has been equilibrated with a trialkyla"f,ne.

WO 91/12342 PCI/US91/010~'' As the ma~ s;~ fatty acylate salts in the C4-Cl2 size range are not generally available C4.. ~ .~ially, they must be ~.nl.f s; ~1, preferably by heating the fatty acid to a t~ ~ alule in the a~plu~ ~, range of 60 80 C and adding one mole of m~ si oxide for every two moles of fatty acid, stirring for a sllffirient penod to assure 5 comrletefli~sQhltiQn(l~ ;Lion) ofthema~.~ ..oxide. Aftercoolingtoroom t~ Lul~, the mq~eS;~ l fatty acylate salt can be stored over a ~lesirc ~nt such as MgSO4 or P2OS to remove all of the water l~luduced in the reaction which is not c~l)u~t~ during heating.
In one mode of re~li7ing the third aspect of the invention, the PCR reagent 10 subsets in the g~ease or wax and in the und~ g aqueous phase are cQmpl~m~nt~ry, so that an ad~lition~l aqlleollc PCR reagent fr~mll~tion need not be pl~ ed for placement above the grease or wax. In another mode, three PCR reagent subsets are l~Uil~l for co...l.le-..--nt~;ol- one in the grease or wax and two aqueous fQrm~ tiQns, one above and one below the grease or wax. The latter mode is especiqlly effective in 15 pl~ ,nLi.lg all PCR reagents from coming together in a single aqueous phase prior to the first cycle of a PCR amplification.
There are many ways to aliquot the wax or gre~ of the present invention into the PCR reaction tubes, whether or not the wax or gre~ cont~ins sllrfnct~nt, polymeric particles, a mq~.~;...n fatty acylate salt, or an em~ ifi~ a~queQll~ solution 20 of a subset of PCR reqg~-nt~ The solid wax or gre~ can be weighed into scpa~le tubes; the small masses involved, generally below 30 mg, require use of a balance with a precision of 1 mg or better, preferably 0.1 mg. However, a much faster and sllffi-~ently precise method is to melt the wax or grease and deliver it vol...n~h ;c~lly with a pOSili~r_ displacement ~lficlu~ adjusted to deliver the ~.. in;n~.. mass giving 25 an effective vapor barrier. Preferably, the miclul~i~l will have a glass dp, as plasdc tips tend to capture wax or grease in a way which reduces precision. An example of a suitable variable-volume glass-dpped posidve~ic~ cen~n~ miwu~i~L is the Micro/Pett~9 m~nl.rs~ d by SMI. If the wax or grease is delivered to a solid surface at room ~.,l~.alule or below, it will solidify as a roughly hemi~pheric~l bead 30 which is easily sep~ t~d from the surface and delivered to a PCR reaction tube (e.g., with forceps). The colder the sllrf~ce, the easier it is to sel)ala~ the wax ~~ grease bead from it. A convenient re~ept~ ~le is a ~licpos~l~le weighing boat, available from many s~;e ~;rc supply ~..~ nies Once the wax or grease pellet and the ~lul,liate volume of an aqueous soludon ûf a subset of PCR reagents has been delivered to a PCR tube, 35 the tube should be capped, heated to a te~ alul~ s~lffic ient to melt the wax or grease for a period of about one minute, and cooled at room lem~.aLwe to seal the wax or grease bamer over the aqueous layer.

''O 91/12342 PC~r/US91/01039 Plastic miclupi~t tips can be rendered more suitable for aliquoting wax if they have been rendered hy~lluphilic as described above for PCR reaction tubes, e.g., by c~ona discl~,e or ~ co~ing Then plastic-tipped air~i~pl ~,em~nt samplers are ade~lual~ly precise for wax delivery. The precision of wax delivery by plastic-5 tipped air-displ~cem~nt samplers also is improved by heating the wax at least 30~C
above its m~lting range. ~ef~ .xl for the a~ ."~t~ aliquoting of wax is a ,lo~.~cessol-controlled multi-tipped air~~ sampler like the ProPette~
lab~l~tc"~ robot, supplied by Perkin _lmer Cetus I~.s~ nl~ .A.~...~ or sem~ ..u~ rapid and precise manufacture of many ll~o~ n~ls of wax beads at a 10 time can be err~d by delivering molten wax at a te-..pe ~ a~ ely 5-15~C
above its melting point by a pl~ ;c~lly driven heated liquid ~ (for example, under regl~lP~ion by an Accura 1 reg~l~t~r for Iwashita F.n~ g Inco.~laled), cQll~ctin~ wax beads on a rotating chilled dlum f~m which they are scraped into a tray after h~l~ ning. For example, the drum can simply be a 2-3 liter glass bottle, 100-300 15 mm in .1;~ ..,t~, filled with ice, rotating at about 1-3 rpm on a culture bottle roller (Wheaton Insll ulll~,nb Inc.).
.Although the previous d~ . ;pl ;on has ç. . ~I h~c:7~ the creation of wax or grease bamers to enable the se~;gd~on of cc~mrlem~nt~ry PCR reagent subsets prior to thermal cycling, it must be realized that wax is such an improv~llh,nt over the mineral 20 oil vapor barrier co~ ly used in PCR that the modes of pl~ g and c~"ni~g a wax barrier desc, ;be~ above may serve well in PCR re~!ion~ where there is no desire to S~ ;alC~ nl~ before thermal cycling. Then the wax, all PCR re~g~nts, and the test sample may simply be mixed lo~lh~,r in a lcac~on tube, ~ f~d~bly within several ...;~..,t~,s of starhng the~mal cycling. As soon as the wax melts during the first cycle, the 25 wax will form a vapor barrier to block solvent e~àpulali~n. When the reaction mixture cools at the end of thermal cycling, it will form a solid bamer to reduce the chance of ull~vatlt~,d PCR product dispersal, a barrier which can easily be ~nch~t~d by a ll~lu~ for PCR product withdrawal. I~ullll.,llllul~, the art of forming and using wax or grease vapor bamers in con~A.n~ ~ with hyd,ul,hilic inner s~lrf~ces or with 30 inClll~ion of nonionic s~ r~ lA~I in the wax or grease, is ~ clc!se~ herein in a manner en~bling adaptation to non-PCR conle~ls, which benefit from any of the advant~ges listed herein. The major ~lj..~l...~ ..l is of the mass of wax or grease to cover completely the e~osed surface of the ~ueou~ co,~ -~nl in the yl~,fell~d cont~ine,~.
Many of the advantages of the present invention can be achieved simply by p ~ ~ging subsets of PCR reagents in difr~l~nt c~nLAin. . ~. In a p cfell~d embo~lim~-nt, ~is aspect of the invention is a kit dlat comprises (a) ûne tube that co"~ns a pair of plil,~ , a nucleic acid polymerase (preferably ~ag. polymerase), and one or more WO 91/12342 PCI/US91/0103'' ~750~
- deoxyribQnucleosi~e ~l;yho~yhat~ s (yl~felably dATP, dGTP, dCTP, and dl~P) in a s~ le buffer, and (b) a tube that C4~n;~ MgCl2 (preferably in solution). The kit can also cQmpri~e h.~ll u~;~ns for call~mg out the PCR process with the kit co~ onenl~.
In particular, the m~ c~ns will ~les~ihe how to mix the co..~ of tubes (a) and (b) 5 with a test sample. In the most pief~.~d e ..b~l;...-- .l, such a kit co.~ es (a) one tube that conl~ins 50 111 of solutior~ ~llyosed of 1.15 uli~,lul"oles of Tris-HCI, pH 8.3;
5 micromoles of KCl; 17.25 picomoles of each primer, 21.6 n~l~o~ s of each of the four deox~ibon.lclP~side l~iyh~tk~ s. and 2.875 units of Ia9, polymerase (PECI);and (b) one tube of 8.05 mM MgCI2. Typical insllu~ions for this kit would tell the user first to layer 50 111 of the MgCI2 sol~tion onto the top of the solution in tube (a), then to add two drops of mineral oiL then to add the sample DNA in a volume of 2 to 40 ~1 on top of the MgCI2 layer, and to place the sample in a PECI TheImal Cycler as soon as possible (under 30 ~--;n~lt~,S).
In yet another ~.Ibodi~nl of the invention, a gel or other matrix co1-t~;..i--g one 15 essenti~l PCR reagent, i.e., ~ag, yol~ se, is layered onto the other co.~ onenls of the PCR ~ ul~i (except sample), and the gel or matrix is co...l osecl such that, upon sample ~1~1ition and h~ ing, the gel or matrix melts and so l~CO~ es a cc.. ~pl~te PCR llfi,~ ,. In one emb~l;..~ nl, the gel is agarose co~ inil~g ~g, polymerase.~hhoU~h this r~ has fc~usod on improve,ll~,nl of PCR pe.Çu n~e7 the 20 invention is ap~' ~ P~le to other modes of en~ma~c repli~ti~n of nucleic acids, such as the ll~ 1 ;on-based ~nrlifir~tion system (Kwoh ~ ~1., 1989, Proc. ~a~l- ~Ç~. ~i.87:1874), ~ Ilpl;r~ ;on systems based on nucleic acid ligase (Wu and Wallace, 1989, C.F l~nll l;-~C 4:560, and R ~ g~!,l ~ j~.~ 1990~ Gene 89: 117), and ~rlifir2tion ~t~,llls based on . ;1~ e H cl~a~~~ of a DNA-RNA-DNA probe ~nnP~1F~d to a 25 nucleic acid target. Ch~,.. t . ;ctir Ç~UI~S of an in vitro nucleic acid ~mrlifir~tinn or detection system which would ~ ~,e improved sl~;r~ ;~r through ~)pliCdl~ll of the invention are the following:
(a) a nucleic acid primer or p~obe, usually an oligonuc!~P4ti~1e, must be ~nnP~lF~ to a target nucleic acid sF~uF~ Ce (b) the .-~ .-, or "stringpnt"~ t~,m~lalule for such ~nn~P~ling must lie above ~mbient t~ll~la~ , (a~ ,Aimately 20 30-C);
(c) a catalyst, usually an enzyme, must act on the ~nne~ l nucleic acid complex;
(d) the catalyst must be active at the strin~Pnt ! nnP~ling t~ a~ and (e) the catalyst must retain ci~l;rlr~." activity at ambient t~ a~ , or the most convenient t~ a~ul~ for mixing re~ct~ntc 'O 91/12342 PC~r/US91/01039 Under these ~ n~, a nucleic acid ~pliration or det~cti~n r~a~;~n is vulnerable to the gen~lion of s;d~p,~lu~ when the pririner or probe anneals to non-target sequ~nces under ~ iS~ t~ure and solvent c~nrlibon~ or when the signal~epe-ndent catalyst operates even in the ~ e of target se~uçnce; and the 5 present in~en~ion l~duces such side reactions by allowing reactant sc~ on before on and reactant mLsing when the b~ , o~ has risen to sh ;.~g. nl levels. In thiscase, one needs to form~ te the wax or grease so that it does not melt at least un~l a~ at~,ly ~ ;l ;onC have been reached. More generally, ho~ e., aspects of this invention may benefit any aqueous .~li~n where a vapor barrier is 10 needed to ~~-;;-f solvent or other reagent ev~r~tinl~ and where there are fimction~
advantages to vapor barrier solidit,v at lower t~ es and fluidity at higher h~ alul~,S.
From the above desc~ and the following e~les, one of or~inary skill in the art can ~ at~ the many diverse aspects of the present invention as enco.~p~sed by the following claims.

FY~ ?le 1 F.ffect of ~C12 S.o,~ .t;~ n on PCR Re~e,nt Sto~s~e St~hility Two ll~lul.,s ("pre-mixes") of PCR reagents were prepared in deioni7f~ water and stored fro~n at -20 C in 50 ~ iq~lotc in 500 ~ ppel d~f llfi~ufuge tubes.
Premix A co~t~inf~l 0.055 C~l1L~11C unit/~l of Th~ lc~ ti~llc ( ~) DNA
,olyll~w~ I cloned and CA1~1CSS~I;1 in E. ~QIi (AmpliTaq~ from Perkin Elmer Cetus InSIIULU~ S); 0.11 mM of each of the four cc.. ~n dNTPs (sodium salts from Pharmacia); 0.33 ~lM of each of two PCR primers ~eci~ed to amplify a 239 or 242 nucl~o~ide sequence of the HLA DQac gene (Saiki ~ ~., 1989, ~. ~
25 II~ 86:6230-6234); 22 mM Tris Cl, pH 8.3; and l lO mM KCl. Premix B cont~ine~1 the same cc...~ as Premix A but also c~ fd 8.05 mM MgCl2.
At various times over a five-day interval, four tubes conl~ g each of the ~le.~ ,S were thawed and placed in an airhght screw-capped plastic cm~t ;nf l at room t~ v-c. At the end of this interval, the tubes were treated in the following ~
(a) 50 ~11 of ~f ;oni,~d water were added to each tube col-l~ ing Premix B; (b) 50 ~1 of 8.05 m~ MgCl2 were added to each tube conl;~h~h~g Premix A; (c) two drops of minPral oil (Sigma ~hPrn:~~l Co.) were added to each tube; and (d) 0.3 or l.0 ng of pllrifi~ human ~e.-.J~ DNA from the human cell line WTSl in 10 ~1 of ~l~Pi<!ni water was added to each tube.
The tubes were capped and immPAi~tely placed in a Perkin Elmer Cetus InSI,UIlh~ TheImal Cycler plo~ll~l~l to mn 32 cycles of the following sequence:

WO 91/12342 PCr/US91/0103'' 94 C for 1 minute, 60 C for 30 ~'~'Q~ , and 72 C for 30 second~ with the fastestpossible thermal st~pping and a 7 minute ~ on of the last-cycle 72 C inrllb~tion.
After this PCR alnplir.r~ the a~ ;rl~l DNA was analyzed by agarose gel el~l.uphul~i.is at room t4lll~a~ in a gel co..l~;ning 3.0 g NuSieve~!9 agarose and 1.0 5 g of Se~K~n~ agarose (FMC Coll,o~ n), and 100 ml of TBE buffer. TBE buffer cont~in~ 0.089 M Tris borate, pH 8.3, and 0.025 M Na2 EDTA. The elecl,~horesis running buffer was TBE. The separation was run at 150 V, al)plu~ly 50 milli~ w,.~ s, for 2 hours. Each gd sample slot ~l~in~ 10 ~ of PCR reaction plus 2 ~1 of 25% Ficoll 400, 0.25% hu.nl-kf,nol blue, and 0.25% xylene 10 cyanol. After cl~LI~hûlt;.is, the gel was stained in 0.5 llg/~ ethi~ m bromide in TBE buffer for 15 n-inules and ~ ed in H20 for 15 l..in llGs The ethiAi~lm-stained d~t.oph~lic bands of DNA were vi~ li7~d on a 300 nm ultraviolet t~n~ , . The tubes cn ~l~in;~g Premixes A and B had beent~ d to four dirr~ ~l;nn intervals at room temperature before running 15 PCR: 1.5 hours, 6 hours, 1 day, and 5 days. All of the Premix A sa,llples, except those int~nb~ted 1.5 hours at room ~,ll~;~,alule before adding 0.3 ng of human genomic DNA, showed the e~ ed 240 base-pair PCR l,r~luc~ band and only trace ~i...O.~.L~ of primer dimer. Only one of the Premix B s~mrles, incub~ted 1.5 hours at room l~m~lalun, before adding 1 ng of DNA, showed the 240 base-pair product. The 20 PremixB s~ "les showedillelca~ingi~..u,)~ of primerdimerandprimeroligomers, directly related to the time the samples were in~u~t~ at room t~l~lalule before PCR.
The data showed that even relatively short ~ J'~ ;onc of compkte ~iAlul~,S of PCR
~,nls at room ~ .qw,- l~ c before mnning PCR inactivate them. This inactivation may be related to primer dimer accumulation. On the other hand, the PCR reagent 25 subset lacking ma~,~~;.--l~,t~incd full activity after even five days at roomt~,lll~lalulc. The present example also shows the pr~ctiC~lity of ~,Çullllulg PCR when ComF~ reagent subsets are mixed just before anlrlific~tion is star~
In other e~ n~s ~ecignP,d and f-Ye~ute,d sub, tS~ntii~lly as ~lf-C~ihe~ above, Premix A (lacking mA~-.Ps;---n) showed rffi~ent and reproducible amplification of the 240 base-pair HLA DQa target from 0.3 ng and 1 ng of human g~nomic DNA and produced no or barely ~let~ blc primer dimer after 12 days storage at -20~C, 4~C, 25~C, and 37~C whereas Premix B al~pl;rled ~e same ~ ~uU~n~ of target erratically and produced con~iderable ~wullb of primer dimer after 21 or 27 days storage at -20~C.
These same r~ n~ showed that vigorous mixing of Premix A with MgCl2 shortly before amplific~tit)n~ rather than layering of reagent subsetc, increased primer dimer yield and l~luccd specific product yield. In still other similar eA~,i.,~n~, Premix A

~'0 91/12342 PCI/USgl/01039 showed co...~ , storage stability for 2 months at 45 C. Premix B failed to produce ~iet~ct~le amplified specific PCR p,udu~ after several days at room t~

FYSIt~I~k~ 2 Fffect of Co~ ,let~ n~ of I~Cl~ SP~ nn PCR Specifi~it,y All PCR reactions used Premix A as defined in E~ lc 1, and the reaction l~lu~.,s were adjusted to 8.05 mM MgCl2 shordy before ~mrlific~tion. The tubes con~ g pre-mix were stored frozen until shortly before use. The test sample c~ncic~d of 0.1 ng/~l p.lrifi~ DNA from the RS-2 cell line; 10 ~1 of test sample and 50 111 of MgCl2 were added to each ~ ion tube before carefully layering 50 ~11 of mineral oil on top. Six ~ tubes were set up under each of three con-litit)n~: (a) the pre-mix was cooled to O C in an ice bucket; 50 ~ of MgCl2 was carefully layered on top; 10 ~1 of DNA was carefully layered on top of the MgCl2; (b) layering was done as in (a), but the pre-mix was at 30 C; and (c) the DNA was layered on top of the pre-rnix at 30 C, followed by the MgCk.
Tmme~ tely after set-up, all tubes were "~sr~.,~ to a Perkin Elmer Cetus II~S~u~ b Therrnal Cycler and ~ub;cct ~1 to the following ~lUE;lall~: 1 minute at 98 C~
30 seconds at 60 C, and 30 s~ lc at 72 C for 2 cycles; 1 minute at 94 C, 30 secûnds at 60 C, and 30 se~on~s at 72-C for 35 cycles; and a final 10 ~ u~ ~ at 72-C. Agarose gel ele~vpl.~,~sis and eth~ um s~qining were ei,se..l;~lly as desç~ihed in Example 1.
All ~;lion tubes showed the ~ - ~;t~ 240 base-pair HLA DQcc product band, but layering e~nAiti~n (a) gave less ~ ~u~ than cQn~litinn (b) which gave less product than c~nAition (c). Layering c~u~ jl;nr~ (a) gave more primer dimer than cc-nr1itic)n (b), which gave more pnrner dimer than c~nditinn (c). Most ci~i~cqntly, layering c~ ntliti~nc (a) and (b) gave a seeond no~ ; r.e pl~lucl closer to primer dimer than to ~ ;r~c pl~uCI in m~leclllqr weight, which was eompletely absent with layering eondition (e). Similar eA~h,.~en~ in whieh no DNA was added shûwed none of the second nons~ r~c pl~lu~;l, sug~ ;ng that it results from n-is~ ;ng of human gt~nnmic DNA rather than from primer oligom~ri7qtion. As this ...;c~., ;n~ g reaction must oecur before DNA ~leniqturq-tion during the first PCR cycle, its eYictcnre implies 30 that the human g~"~OIlliC DNA of the test s. mple must be signifir~ntly single-stranded.
When the ele~;llu~,horetic mobility of this non~ ;r~c product was co~ Jd,~d to those of fr~E~nentc from a ~III digest of ~X174 RF DNA (si_e standards from Bethes~
Research Labu,~,ies), the no..~ ;r.~ ~loiu~l was seen to be a~,uAi,,lalely 91 base pairs in si_e. Primer dimer was a~l,lu.,;.~-A4~,1y 68 base pairs in si_e.
This ~ ~pf ;~ l cc~nrl~ d the value of segregating m~.. e~iwll from the c~.npl~ ,n~.~/ PCR reagent subset until the actual start ûf PCR thermal cycling.

WO 91/12342 PCI /US91/0103"

Int~.~,osi~,on of even just a 10 111 layer of PCR-reagent-free test sample ~l.. ~n the two reagent subsets served to reduce plimerdimer f~nnq~ n and prevent a ...;~.;..-;--g-~epen~ent ~ -.pl;r~ ;Qn of l~on~ ;rle ~loducL Surpnsingly, layering at 0~C gave more p~imer dimer than layering at room t~,~lalule. Taq DNA poly~l~se I appears to catalyze primer dimer formation wen at O C.

~am~ 3 F.ffeCt nf ~g. ~'m'9~. nr Wsl~ Vs~l~nr Rsrri~o.r PCR reagent Premix A and stock 8.05 mM MgCl2 were p~ ,d as ~es~ibed in Example 1. The human genomic DNA used as a test sample was ~--- ;red from thecell line HL 60 and f(~ ls-t~ in water at a conl~-ul~ alion of 0.3 ng/~l. To PCRreaction tubes con~q-ini~g 50 ~11 of ~remix A were added 40 mg masses of the following waxes or grease: ps-rarlsct (Monoject T~ sl~ ;PS), 58-60 C melting ~al~fin (Aldrich Chemir~l Co.), octacosane (Aldrich Chemical Co.), Vaselhl~,~ Petroleum Jelly (Che~seb~ ughPonds),r~ L_l.y~;tol~h~h~ 9t. (LipoCh~m:-Alc)~andBeSquare 175 (Calwax Cul~laliOn). Several otherreaction tubes ConlAinil~g 50 ~11 Premix Areceived 75 ~1 of light mineral oil (Sigma Chemical Co.). All tubes were capped and ,ul.~3 in an 85 C water bath for 90 120 se n~lc (until the wax melted into a layer over the premix), then cooled to room temperature.
On top of each wax, grease, or oil layer was added a ~ UlG co~ g 50 ~11 Of 8.05 mM MgCl2 and 10 111 of 0.3 ng/~l pllrified human gennmil~ DNA. The mixture added on top of oil i~ ,h~t~ ~d the oil to join the premix. All tubes were Ç~ xl to a Perkin Elmer Cetus Irl~hu~.~ Thermal Cycler already heated to 90 C.
After 90 se~?n~c at 90 C, 35 cycles were ~ vte~l acc~lding to the following pl~o~
30 5C~O~ at 95 C, 30 se~ol-AC at 55 C, 30 s~ nrlc at 72 C, with a final 7 minuteeYt~n~i~ n at 72 C. Thermal ramps ~t~. ~n temperatures p.~,e~eA as rapidly as possible.
PCR product was analyzed by ethi~lillm-stained agarose gel ele~ll~hc,.~is essc.nl;~lly as des~ibeA in Example 1. All of the re~ction~ with the wax and oil vapor barriers listed above gave the specific 240 base pair PCR product. Paraplast, OC! -CO~~,~f, and penta~ ol tetr~l~en~e gave specific PCR product in com~ a~le yield to mineral oil. Various waxes gave pnmer dimer in yields ranging from none to heavy, wh~leas mineral oil gave a small amount of primer dimer. ~ ;n~.,y ..n~ of the same design with certain other waxes, such as eic4s~ne (Aldrich C.hemic~l Co.), ~ iCo~-G (Aldrich ChPmi~l Co.), t~t~.~cos~,ne (Aldrich Ch~mi~l Co.), 35 and c~ lba wax (Calwax C~ alion) gave no PCR product. However, later e~ showed that ~lro~ ce of wax layers in the mass range of 40-50 mg 'O 91/12342 PC~r/US91/01039 33 2 ' was erratic; ~ ;...es the MgCl2 and DNA ,.~lul~, would easily fall through the ~melted wax and mix well with the premix but sc..n--l;..~,s would not. Th~lerore, single ~cces~rul or lmc~ rul reactions with such large wax masses predicted neither reliable qt~lifir?tton nor ullirw~ unsuitability. Fcr example, later e~~ . ;.. .l~
5 showed that eiGosqne worked as well as Paraplast and y~rl,~, which gave yields of both specific product and pIimer dimer co.n~ hlc to thos~e with a mineral oil vapor baTrier. Cetyl pqlmitqte (Serva Bi~h....;~alc) and Ultraflex (Calwax C~ ~ion) also se~ved as liquid barriers yielding useful ~ 0!~-~15. of spe~tfic ~ u~;l and only low ~ ~,o...~t~ of pnmer dimer. MineIal oil was mixed with l,C ~rr..~ and with pqr~pl-qct at 10 c~ nce~ a~ions up to 25% (by mass) to give liquid barriers which gave spe~tfi~
amplir~ n as good as mineral oil alone with lower yields of primer dirner. Theseul~,s gave no higher PCR s~-;r~ or yield than the waxes alone, but they were S{lllh,~. hal softer and easier to ~ eh~ with sampler tips used to withdraw the PCR
product for de~ lloph~ ~ic analysis.

F~s."~ 4 M;..;...;,;l~ th~ MstCC of a W~x V~or RSttTier In Example 3 and related é~ Ic, a 40 mg mass of wax was layered over 50 ~1 of Premix A; and a 60 ~ ul~i of MgCl2 and DNA was placed above the wax was so that the final ~lleO~tC volume under the wax was 110 ~ tholl~h these 20 ~ ;l;es led to complete coverage by the wax liquid ba~rier before thermal cycling and complete coverage by the wax vapor barrier during and after thermal cycling, theres~lting wax layer was so thick that post-PCR lJf nf~AI;~>~ by a ~ tip was i"co,~e,~ent and l~ui~,d close control. The ~ ,S~>Ul~, on the lnicl~i~l needed to break through the wax often led to sudden ~"~ t~d~ion and spur~ng of PCR reaction 25 ~lu,e (inr~ ine arnplifi~ nucleic acid) past the mi;lupi~l tip, ~t~.,~ially c~.~ ;fle the lab~ldt~l~ em~ilo,~nl with PCR ~l~Jducl which could back-later A~ lir.~r.~ -s F.~l~. ;...~ l~1~ to test the ...;~ .. mass of pdl~rrln c~ ing completely 100 111 of water showed that ~. ~n 30 and 35 mg of wax were needed for collll,lete coverage 30 as a liquid bamier, ~lthough vapor bamier effectiveness (re~ucti.~n of water evaporation) a~d to be coll~plet~ .~n 25 and 30 mg. The PCR reaction tube was a standard ClAl~500 ~ luc~ce.,llirugetube~ r~ dbyCoStar. Thesee~ did not require thermal cycling, but only visual ~ AI;on of wax layers under low ~..~,-;r,cq~ion to detect holes, followed by heating of the closed tubes to 99 C for 10 35 ...;..~ ,s befoqe ~ e the mass of water col-d~ on the walls and in the cap of the tube. The ~uCtul~ ne4uil~ nl for so much wax derived fnom the fact that the W O 91/12342 PC~r/US91/0103~
2 E~ 5~
wax, like mineral oil, formed a concave-d~w,l-.dld menic~uc with the aqueous co~ .l~nl below it and a concave-upward ... ni~ c with the air above it at the time that the layer was forme~ The.~,f~"e, to get co..q.l~ t~ coverage at the center of the tube, a much thicker layer was needed at the wall of the tube. The focus of efforts to render 5 the wax layer thin enough to be practical rested on co..q~ n~ which reduced the depth of the .~u nicri creating a more uniîc~lLu ~ ul ;.~n of wax across the top of the a~ueo lc cc""~ ~nt, in the Ih;nn~ ~l hyer giving complete coverage. Pa~rm was the wax used in all of these efforts. ~lthongh other waxes must show the same cerni~ e behavior as pa~affin, their ~ e ~ rnight differ SOLIJ ,-. Ildl. e Tncl..~:on in the p ~,Ir~n of 1% Brij 52 (diethylencgl~col ..d~noce~yl ether, Serva Fine Biochemicals), Brij 30 (triethylene~l~col monols~lryl ether, Serva Fine Biochemicals), or pol~ Ih~lene-9-lauryl ether (Sigma C'ht~m;~l Co.) reduced the minimsl wax mass for complete coverage from ~l~,. ~n 30 and 35 mg to ~l~. ~n 20 15 and 25 mg, but llliCI~)i~l tip ~n~ ion was still ~liffi~ult Tnrlmion in the wax of 20~400 mesh poly~ ine-divinyl~4n f ne beads (BioBeads SM-X8, BioRad Laboratories) at mass percent of 10 or 20 l~lucod the .n;n;.. , effective mass of wax to beh.~n 25 and 30 mg. More benrfiriqlly~ ~ l~i~l tip pen~ ;on was effortless, and the wax brvke away wilhoul clc~ing the ~smrl~ tip. Bead cc~n~e ~l~a~ions of 5%, 30%. and 40% did not give complete coverage at 30 mg of wax. Although 10%, 20%, or 40% (by mass) of Vaseline~ Pet~vleum Jelly in y~ n was not tested for effect on the minimql mass of wax needed for cc~ k t~'~ coverage, at 40 mg of these IniAlul~s (svl~.hal above the robable ~--;~ --), rnic,v~,yel tip ~..~eh~ n was easy because the wax was sv~..llat ,oft~ned. IIo..~ , the wax tended to clog some sampler tips.
25 Tnr~ ?n in a mere 10 mg of y--~lr,n of a 3 mm ~l;A~ te~ circle of polypropylene monofilsnu n- mesh (~uy~llcA~ silk sc,~.~ing fabric; Tetko Cv~yol~ûn) weighing ayylvA;~ t~ly 0.5 mg gave con~ cv~erage of 50 111 of water in an ~ ~,roS~
polypropylene ". crvc~ iruge tube. Repeat of this e~ t with 7 mg of wax didnot quite give cv,n~lete coverage. Although a Swl~ . I,at larger mass of wax would be 30 needed tv cover the 100 ~1 of water used in the previous tests, that Amollnt would not nearly a~ luach the 30-35 mg needW in the absence of mesh or surfactant.
To test the effect of coating PCR reaction tubes with sllrfartAnt, 500 ~
mi~;,vcer,lliruge tubes were incubated fvr 30 n.;n..t s at room t~lll~,.alul~, CQ~ g 5% solutions in l-l/~u~anol of each one of SiA ~... ra~ "~ polyoxyethylene-9-lauryl etha, Span 40 (sorbitan ",~ ~qlmit~tç Sigma Chem~ l CO.), Brij 30, Tween 85 (polyc,A~lhylene s~,l,ilan trinl~t~; Sigma (~h~mir~l CO.), Span 80 (sc.,l,i~n monooleate; Sigma Ch~mir~l Co.), and Brij 52. After d,~ age of the ~ -- r~

soluti- n~ from the tubes, the tubes were air-dried. Various masses of p&arrli~ (7.5, 11.3, 15, and 18.8 mg) were layered on top of 50 ~11 of 0.06 M KCl in H2O in each tube. After the wax layers had h~ d and were exs~nin~ visually for holes under low mq~-ir;r -';Qn, they were covered with 50 ~1 of a 0.1% solution of methylene blue 5 in H20. After the tubes were capped and stored for 3.5 days at room ~ ~laLul~, they were e~ ~l visually for signs of dye leakage from the upper aqueous layer into the lower one. Then they were ~,~d for two ~;n~t~s in a 95-97 C water bath, allowed to air-cool to room ~ , and ~ L~ ~3 visually for signs of holes inwax covering the 100 111 of c~..~ d aqueous layers. During h.osting, they were 10 observed for the timing and cc,~lJlet.~ess of dye ~ )~ ;on of the melted wax and mixing with the lower aqueous layer.
~ hhough two sulr~lant~ (Tween 85 and Span 80) gave greatly improved ~lrv-~ nce over a control tube which was ~ o~ particularly in providing cc,~ t~, coverage of 50 ~1 of aqueous solution with only 15 mg of wax (no holes and no dye leakage after 3.5 days), only Tween 85 gave complete coverage of 100 ~1. Two other ~ . r~ . (polyo~ lene-9-lauryl ether and Brij 30) gave partial impro~ nt over uncoated tubes; no holes were seen in wax covering 50 ~1, and dye ~n~,tl~onbefore heating did not occur ;,,,,,~ 4 ly, but l~uil~d several days of incu~-s-tion In this test, Span 80 was judged ~uestir~ns1~le on a dilrw~ ground; it was the only20 coating which ap~ to impede ~.-~.tlah~n and mixing of the upper aqueous layerupon heating to 95-97 C. Th~lefijl~" Tween 85 provided special benefit in co...~ . ;con to the other surfactants tested or to no co~ting~ g~ly l~]~cing the mass of needed for co- . .l.l~ h Cu~w~g~, of 100 ~1 of water from ~l.. ~n 30 and 35 mg to a~l,lv~ t~,ly 15 mg. I~ , the l5 mg layer of wax covering an 100 111 25 aqll~o.lc colll~)&~Rnl was very easily pelh;hal~d by a miclu~J;~l tip. IIv..~ l, it is e l,e.te~ that other surfactants not yet tested will meet or surpass this ~Ir~....An~e, as those tested l~,pl~ SCI~t only a small fraction of the hundreds available co-.~ ..;ially.
In a further test of Tween 85~oated reacdon tubes, 7.5 mg, 11.3 mg, and 15 mg ~,alarrln layers co ~ ;nil~g 0%, 0.34%, and 1.2% of Tween 85 were used in an 30 ~ f ;n~ just like the s..~ r~ coating screen just ~le~ibe~ This time the mass of water ~liCtilled onto the upper walls and cap of the tubes during 10 ...;n~les at 99 C was measured This last critefion showed a marked benefit from either Tween 85 con~enl.alion in the wax (Tween 85 also coating the tubes). S~r~-! --t also helped to acl~/e col~let~ coverage of 50 ~1 of aqueous solution with only 7.5 mg of wax, but did not allow complete coverage of 100 ~I with either 11.3 mg or 7.5 mg.
Stdll further tests of Tween 85, using a similar ~ .. pf . ;. . ~ 1 design, showed that 1% Tween 85 in 56-61-C melting l,~uarrln worked a~l~ lely equally well in WO 91/12342 PCr/US91/010~
207~a50 36 Tween 85-coated and ..ncwl~ tubes. Il~ r, this test did not include storage for several days or actual PCR qmrlificqti~n Both co~ ;on~ gave better co.~ug_ than the use of Tween-85-coated tubes without Tween 85 in the wax, and much better coverage than the use of un~l~,ated tubes 5 without Tween 85 in the wa-A. ~c~...l;~ to the dual criteria of easy .lliclu~
pe~ ~io,. and complete coverage (no holes in the waA and minimql eva~ ~.oll of water from reaction ~AIUl~ during thelmal cycling), the following wax masses were found to be pl~f~l~d for the z~s~,;~ d~,.lying watervolumes when pa,~rl.l CQI.l~ ~ 1% Tween 85: 8 mg with 25 ~1 water, 12 mg with 50 111, 16 mg with 75 ~1, 18 mg with 100 ~1, 22 mg with 150 ~1, and 26 mg with 200 ~1. These masses are several mg higher than the n~;n;-n.-.-- effective masses, but help to assure that all tubes will p~ .. well, as there is certain to be some ranAa.. ~r~ in both the mass of wax delivered and the res~llting performance.

F.~n~lr S
F-ffect of T~oved W~qY nn PCR Arru~lifi.~q-ti-)n Tween 85 was dissolved in l-pl~al ol to a cQnce.~ ~.on of 1% by mass.
Ap~ tely 0.5 ml aliquots of this Tween 85-propanol solution were added to 500 licl~xenl. ;ru~e tubes and incubated at room ~nq~~ ; for about 30 .~ ,s before A~ining the tubes with a glass Pasteur pipet. The tubes were dried under vacuum (22 inches Hg) at room ~ n~e for 30 .. ;.. ,t~,s Tween 85 was dissolved at 70-90 C in 56-61-C melting paraffin (Aldrich Chemical Co.) to a co~-~e-~ of 1%. rou.~n mg ~ u~!tc of 1% Tween 85 in ~ were d~ ,d with an SMI MicrolPettor from 70-90 C melted wax onto a pol~ lene wdghing boat sitting on ice. The individual 14 mg pellets of h~n~ wax were deli~ ;d individually to s~ ale Tween 85 coated 25 reaction tubes. Fifty ~ uots of Premix A (FY~mrl~ 1) were added to the wax-c4nl~ini.~ tubes, which were incubated a~ )~ately 2 ...;~ t~,s at 7~80 C and allowed to air cool to room temperature. All wax layers were free of holes. On top of them were added 60 111 of ~e of 6.71 mM MgCl2 and enough pl~nfie~A human genom;~ DNA to contain 30 g. ~ ;c copies (Q 1 ng).
These completed l~ac~n tubes were ~ubj~t~d to the following amplification plU~ llll in a Perkin Elmer Cetus Il~s~ Thermal Cycler two cycles of 1 minute at 98 C, 30 ~con-l~ at 60 C, and 30 seconds at 72 C; 35 cycles of 1 minute at 94 C, 30 se~on-ls at 60 C and 30 sec~n~l~ at 72 C, followed by a 10 minute incubation at 72 C. All thermal ramps bet~. een t~ ul~s were ~ r~,l-lled as rapidly as possible.
35 Fthirlinm-stained gel elec~ h~ ,is was p~.r~-~d e~ .l;~lly as in Example 1.
Sixteen replicate .~ .~I;o~s were run as just ~les~ibe~ Another six tubes contained mineral oil instead of wax; in them the 60 ~1 of DNA and MgCI2 mixed ;...nkYl;~r~ly with the 50 111 of Premix A upon ~ ition to the tubes. One more cont~ol tube used wax with no added DNA and one used oil with no added DNA. Gel cle~ ul,hulesis showed that fifteen of the sixteen wax-covered ~ l;r,r~ nc g~n~,l~l the e~t~A
5 240 base-pair ~ ;r.r PCR product and cignifir~nt amounts of pnmer dimer. Three of the fifteen showed si~;r~l~a~ less of the specific IJludu.;l than did dhe others. All six oil controls showed s~.ml~,.. hal but not gready higher yields of spe~ific product than did the ~vcces~rul wax co.el~d ~ ;r~r~ations~ but they also showed the 91 base-pair mix-pnmed l~o~pe~;r~r~ Alu~ ec~il~ in Example 2, completely absent when wax was 10 used. Omicc;on of test sample DNA resulted in a n~rmal yield of primer dimer and no ~etect~'e 240 base-pair or 91 base-pair pr~l~l~;l. Wax gave solll~.hal larger pnmer dimer than did oil, and sol~hf~ hat higher yields of primer dimer than did oil.
This ~ cnl showed !~ce~t~ y l ~ u;~le PCR ~mpl;rir~;o~ when 14 mg of paraffin cc lit; ;-~ g 1% Tween 85 ,~laces 100 ~1 (applu.~ t ly 80 mg) of15 mineral oil and sen~es to se~ ga~ MgCl2 and test sample f~m the ~ inil~g PCR
reagents until the first ~ ;r~r~;oll cycle. This ~ ~q~l;r~a~;on was Si~;r~rAn~ly more se.l~ili~_ than those in Examples 1-3, which started with at least 100 g~.-o.~ copies of HLA DQo~ DNA.
Similar e,.~~ in which the mass of wax was ~3ou~le~ from 14 to 28 mg 20 showed much l~luced yields of specific ~l~lucl and almost n~nal yields of primer dimer. Similar ~ .l~.;...~nl~ (with 14 mg of wax) in which test sample was added to premix before wax layering and only MgC12 was plaoed above the wax seemed to give SOll~. hal higher and more ,~ r;~lc yields of s~ifi~ product and lower yields ofprimer dimerplobably because the test sample spent more time at high l~ ...~ .~l...
25 during the first one or two cycles. Similar r~pe ;~ in which the 60 ~1 of test sample and MgCl2 were placed below the wax and the 50 ~l of premix were placed above also ~ d to give higher and more ~ ~lucible ~ ;r.~ pl~du~;l yields and reduoed primer dimer yields. These last reactions also gave a more conlrlio~te~ and variable primer dimer pattern, more like that seen with oil vapor barriers. These 30 variations on the normal way of using Tween 8s~N-l~;ning wax to se~al~ Mgck and test sample from the other PCR reagents in Tween 80 coated tubes suggest that ? 1.l;1 ;on~l impro~ _m~ .lb may result from further reduotinn in the wax mass and by ing the wax melting point so that the rea~ants mix more rapidly after the first the~mal cycle is started. These ch~nges and other simple ones, such as prolonging the 35 du,~ion of the first-cycle dend~ion se~..~ ..l (-;ull~ ly 1 minute at 98 C) can be used to improve first-cycle DNA d~alul~ion and thereby increase yield.

Claims (52)

In the Claims

1. A PCR reaction tube comprising a container and an aqueous mixture comprising all PCR reagents except a magnesium compound, wherein the total magnesium concentration in said mixture is less than about 10-4M.

2. The reaction tube of Claim 1 also comprising an aqueous suspension or solution of a magnesium compound, wherein said magnesium-containing suspension or solution is present as a separate layer substantially unmixed with the aqueous mixture of PCR reagents complementary to magnesium.

3. The reaction tube of Claim 2 also comprising a test sample containing nucleic acid.

4. The reaction tube of Claim 2, wherein the two layers containing, separately, the magnesium compound and the PCR reagents complementary to magnesium, are separated by a third aqueous layer containing no PCR reagents, wherein all three layers are substantially unmixed with one another.

5. The reaction tube of Claim 2, wherein the two layers differ in density by at least about 0.2 g/ml, and the lower layer is the denser of the two.

6. The reaction tube of Claim 4, wherein the three layers differ in density from one another by at least about 0.2 g/ml, the lowest layer is the most dense, and the top layer is the least dense.

7. A PCR reaction tube comprising a container, a first aqueous mixture comprising a subset of PCR reagents, a second aqueous mixture comprising the subset of PCR reagents complementary to that in the first aqueous mixture, and a layer of grease or wax which completely separates the two aqueous mixtures.

8. The reaction tube of Claim 7, also comprising a test sample containing nucleic acid, wherein said test sample is mixed with one of the two subsets of PCR
reagents.

9. The reaction tube of Claim 7, wherein one of the two subsets of PCR
reagents is a magnesium compound.

10. The reaction tube of Claim 7, wherein said grease comprises white petrolatum.

11. The reaction tube of Claim 7, wherein said wax comprises a substance taken from the group consisting of paraffin, eicosane, octacosane, cetyl palmitate, pentaerythritol tetrabehenate, Paraplast TM, Ultraflex TM, and BeSquare 175 TM.

12. The reaction tube of Claim 7, wherein said grease or wax is combined with an oil, wherein the resulting mixture possesses substantially the hardness of the grease or wax.

13. The reaction tube of Claim 7, wherein said grease or wax is combined with a nonionic surfactant, wherein the concentration of surfactant in the grease or was is in the approximate range of 0.1% to 1% by mass.

14. The reaction tube of Claim 7, wherein said container consists of a plastic, wherein the inner surface of said container is hydrophilic.

15. The reaction tube of Claim 7, wherein said grease or wax is combined with plastic mesh, wherein said mesh has openings with a maximum lineal dimension in the range of approximately 0.01 to 1 mm.

16. The reaction tube of Claim 7, wherein a line perpendicular to the wax or grease layer lies at angle displaced about ten to thirty degrees from the vertical when the reaction tube is placed in a thermal cycler.

17. The reaction tube of Claim 7, wherein one or both aqueous mixtures also comprise a nonionic surfactant.

18. The reaction tube of Claim 12, wherein said oil is liquid petrolatum.

19. The reaction tube of Claim 13, wherein said surfactant is taken from the group consisting of Tween 65 TM and Tween 85 TM.

20. The reaction tube of Claim 14, wherein said inner surface has been rendered hydrophilic by coating with a nonionic surfactant.

21. The reaction tube of Claim 14, wherein said inner surface has been rendered hydrophilic by plasma etching or corona discharge.

22. The reaction tube of Claim 14, wherein said plastic comprises polypropylene.

23. The reaction tube of Claim 15, wherein said plastic comprises polypropylene, polyethylene, polymethypentene, polyester, nylon, or a fluorocarbon.

24. The reaction tube of Claim 20, wherein said surfactant is taken from the group consisting of Tween 65TM and Tween 85TM.

25. The reaction tube of Claim 20, wherein said coating was performed by contacting said surface with a solution of said surfactant at a concentration between about 0.1% and about 10% by mass in a solvent, substantially free of water, comprising methanol, ethanol 1-propanol, 2-propanol, 1-butanol, or 2-butanol.

26. The reaction tube of Claim 20, wherein said grease or wax is combined with plastic particles, wherein said particles have a density between about 0.6 g/ml and about 1.1g/ml and a maximum lineal dimension in the range of approximately 0.01 to 1 mm.

27. The reaction tube of Claim 26, wherein said particles comprise polypropylene, polyethylene, polymethylpentane, polymethylmethacrylate, or polystyrene.

28. A PCR reaction tube comprising a container, an aqueous mixture of PCR
reagents, and an amount of grease or wax sufficient in mass to separate completely the aqueous mixture from the atmosphere above it.

29. The reaction tube of Claim 28, wherein said container is plastic and wherein the inner surface of said container is hydrophilic.

30. The reaction tube of Claim 28, wherein the grease or wax comprises an emulsion with an aqueous solution or suspension of a subset of PCR reagents, andwherein the aqueous mixture below the grease or wax layer comprises a different subset of PCR reagents.

31. The reaction tube of Claim 28, wherein said grease or wax comprises a solution in wax or grease of a salt containing magnesium and the conjugate base of a fatty acid and wherein the aqueous mixture below the grease or wax layer comprises a subset of PCR reagents different from magnesium.

32. The reaction tube of Claim 28, wherein said grease or wax comprises a solution in grease or wax of a tertiary alkyl ammonium salt of a nucleotide triphosphate and wherein the aqueous mixture below the grease or wax layer comprises a subset of PCR reagents different from any nucleoside triphosphate incorporated in the grease or wax.

33. The reaction tube of Claim 28, wherein said grease or wax comprises a solution in grease or wax of a tertiary alkyl ammonium salt of an oligonucleotide and wherein the aqueous mixture below the grease or wax layer comprises a subset of PCR
reagents different from any oligonucleotide incorporated in the grease or wax.

34. The reaction tube of Claim 28, wherein said grease or wax also comprises a non-ionic surfactant.

35. The reaction tube of Claim 28, wherein said aqueous mixture also comprises a test sample.

36. The reaction tube of Claim 29, wherein said inner surface has been rendered hydrophilic by coating with a non-ionic surfactant.

37. The reaction tube of Claim 29, wherein said surface has been rendered hydrophilic by plasma etching.

38. The reaction tube of Claim 29, wherein said plastic consists of polypropylene.

39. The reaction tube of Claim 31, wherein said fatty acid is taken from the group containing four to twelve carbon atoms.

40. The reaction tube of Claim 34, wherein said surfactant is taken from the group consisting of Tween 65TM and Tween 85TM.

41. The reaction tube of Claim 36, wherein said surfactant is taken from the group consisting of Tween 65TM and Tween 85TM.

42. The reaction tube of Claim 36, wherein said coating was performed by contacting said surface with a solution of said surfactant at a concentration between about 0.1% and 10% by mass in a solvent substantially free of water, comprising methanol, ethanol 1-propanol, 2-propanol, 1-butanol, or 2-butanol.

43. A composition comprising a plastic container containing an aqueous mixture, wherein the inner surface of said container is hydrophilic and wherein said mixture is covered by a layer of grease or wax sufficient in mass to separate completely the mixture from any atmosphere above it.

44. A composition for use in PCR amplification consisting of a mass of grease or wax pellets in the range of about 5 mg to about 50 mg contained within a container suitable for holding PCR reagents and test sample during a PCR amplification in a total aqueous volume between about 10 µl and about 200 µl.

45. A kit for use in PCR amplification comprising one or more containers suitable for holding PCR reagents and test sample during a PCR amplification, a mass of grease or wax sufficient to cover the exposed surface of PCR reactions performed in the containers, or to separate two aqueous mixtures comprising subsets of PCR reagents which are complementary to each other, instructions for how to deliver useful amounts of grease or wax to individual containers, and instructions for how to deliver useful amounts of grease or was to individual containers, and instructions for how to use said containers containing said grease or wax for PCR amplification.

46. A composition for use in PCR amplification comprising wax or grease pellets, mixed with a non-ionic surfactant, wherein said composition melts in the range of about 40°C to about 90°C and wherein said surfactant is dissolved in said wax to a concentration between about 0.1% and about 1%.

47. The composition of Claim 46, wherein said surfactant is taken from the group consisting of Tween 65TM and Tween 85TM.

48. A PCR reaction tube comprising (1) a container, (2) an aqueous mixture of PCR reagents, test sample, and PCR product, (3) a liquid barrier of wax or grease completely covering said aqueous mixture, and (4) a second aqueous mixture above said layer of wax or grease, said second mixture comprising a reagent for reacting with said PCR product.

49. The reaction tube of Claim 48, wherein said aqueous mixture of PCR
reagents lacks at least one reagent required for PCR amplification and wherein said mixture retains substantially full activity in PCR for a period of at least about a week when stored sterile and closed to the atmosphere at any temperature between about 0°C and about 40°C.

50. The reaction tube of Claim 48, wherein said reagent for reacting with said PCR product is an isopsoralen.

51. The reaction tube of Claim 50, wherein said second mixture also comprises a chelator capable of binding to Mg+2.

52. The reaction tube of Claim 48, wherein said reagent for reacting with said PCR product comprises an oligonucleotide with a base sequence at least part of which is complementary to a portion of the sequence of one strand of said PCR product.