CA2102784A1 - Identifying, isolating and cloning messenger rnas - Google Patents

Abstract

A method for isolating mRNAs as cDNAs employs a polymerase amplification method using at least two oligodeoxynucleotide primers.
In one approach, the first primer contains sequence capable of hybridizing to a site immediately upstream of the first A
ribonucleotide of the mRNA's polyA tail and the second primer contains arbitrary sequence. In another approach, the first primer contains sequence capable of hybridizing to a site including the mRNA's polyA signal sequence and the second primer contains arbitrary sequence. In another approach, the first primer contains arbitrary sequence and the second primer contains sequence capable of hybridizing to a site including the Kozak sequence. In another approach, the first primer contains a sequence that is substantially complementary to the sequence of a mRNA having a known sequence and the second primer contains arbitrary sequence. In another approach, the first primer contains arbitrary sequence and the second primer contains sequence that is substantially identical to the sequence of a mRNA having a known sequence. The first primer is used as a primer for reverse transcription of the mRNA and the resultant cDNA is amplified with a polymerase using both the first and second primers as a primer set.

Description

WO 93/18176 ~ 4 Pcr/uss3/o2246 "Methods to clone mRNA".
Background of the Lnvention This application is a continuation-in-part of t~ c~pending application W.S.
Serial No. 07/850,343, f~ed on March 11, 1992.
l~is invention relates to methods of detecting and cloning of individual mRNAs.
The activities of genes in cells are reflected in the kinds and quantities of their mRNA and protein species. Gene expression is clucial for processes such as aging, development, differentiation, metabolite production, progression of the cell cycle, and infectious or genetic or other disease states. Identiflcation of the expressed m~As will be valuable for the elucidation of their molecular m echanism s, and for applications ~o the above processes.
M aIn m al~n ce~s conta~n apprDxim ately l5,CKNO different nnJ~N A sequences, ho w ever, each rn~N A sequence is present at a dif~erent f~equency within the ~ce~. G en~ra~ly, Dn~N As ane expressed at one of tb~s l@vels. ~ few Uabundantn : m ~N As are present at a~H)ut lO,CKNO ~ pies per ce~, abK~ut 3,oK~D-4,CH~D
~; UDntormedOaten~ nnI~N As are present at 3CNO-SCK) copies l~er ce~, ~ d about ll,C~ND
Ul~w-abund~mce" or "~are" mRNAs ale present ~at aF~p~Dxirnabely 15 c~pies per cell. The numerous genes ~at are: ~resented by il~tennediate and low f~eque~eies OI their~mRNAs can be cloned :by a variety of well established techniques (see forexample Sarnbmok etal., 1989, Moleculax Cloning: A
Laoolatory ~lanual,: Second 13dition, Cold Spring Ha~bOE P~eSS~ ~P. 8.6-8.35).
If some knowledge ~f the gene ~sequence or protein is ~ad, severa1 di~ect cloning methods are~ available. However,: if t~ie~ identity of the desired gene is : ~ 25 unlcnown one musl ~be able to select or e~ch for the desired gene product in order to identify the!6'un~own" gene without expending large amounts of ~ime and resources. : ~:
The idelltifuation~:of unknown gelles can often involve the use of subt ctive ordi~ferential hybndization techniques. Subtractive hybridization techniques rely upon the use;~of very closely relate~ cell populations, such t~at diffelences in gene expression will p~marily represent the gene(s) of intsrest. A

: :

2:~ n2~,Qy WO 93/18176 ` Pcr/~s93/02246 key element of the subtractive hyblidization tech2~que is the construction of a comprehensive complementary-DNA (UcDNA) lib~uy.
The constluction of a comprehensive cDNA library is now a fairly routine procedure. PolyA mRNA is pre~ared ~rom the desired cells and the first strand S of the cDNA is synthesized using RNA-dependent DNA polymerase (reverse transcnptase") and an oligodeoxynucleotide p~mer of 12 to 18 thymidine residues. The s~ond strand of the cDNA is synthesi~ed by one of several methods, the more efficient of which are commonly known as Ureplacement synthesis" and UpI~med synthesis".
Replacement synthesis involves the use of ribonuclease H (URNAase H"), which cleaves the phosphodiester backbone of RNA that is ~ a RNA:DNA
hybrid leaving a 3' hydroxyl and a 5' phosphate, to produce nicks and gaps in the mRNA st~nd, creating a se~ies of RNA primers that are used by E. coli DNA polymerase I, or its ~KIe~ow" fragment, to synthesize the second st~nd of 15 the cDNA. This reaction is veIy effilcient; however, the cDNAs produced most o~ten lack the 5' tenninus of the mRNA sequence.
Primed synthesis to gelle~ate the second cDNA st~nd is a gene~l name i~or several methods which are more difficlllt tl~ placement synthesis yet clone the 5' ~enninal sequences with high efficiency. In general, after the synthesis of the 20 first cDNA stIand7 the 3~ end of the cDNA st~and is extended with terminal t~ansferase, an enzyme which adds a homopolymeric "tail of d~oxynucleotides, most commonly deoxycytidylate. This ~ is then hyb~i~ to a p~mer of oligodeoxyguanidylate or a syn~etic fragmen~ of DNA with an deoxyguanidylate tail and ~he second stIand of the cDNA is synthesized using a DNA-de~endent 25 DNA p~lyme~se.
The primed synthesis method is effective, but the method is laborious, and all resultallt cDNA clones have a ~ract of deoxyguanidylate immediately upstreamof the mRN~ sequence. This deoxyguanidylate traet can interfere with ~scription of the DNA in uitro or in vivo and can inte~fere with the sequencing 30 of the clones by the Sanger~dideoxynucle~tide sequeneing method.
Once both cDN~ strands have been synthesiæd, the eDNA lib~ary is constmcted by cloning the cDNAs into an appropriate plasmid or viral vector.

WO 93/18176 ? 1 ~ ',? 7 ~3 4 Pcr/us93/o2246 In practice this can be done lby directly ligating the blunt ends of the cDNAs into a vector whieh has been digested by a restriction endonuclease to pr~duce blunt ends. Blunt end ligations are very inefficient, however, and tllis is not a common method of choice. A generally used method involves adding synthetic S linkers or adapters containing restriction endonuclease recogl~ition sequences to the ends of the cDNAs. The cDNAs can then be cloned into the desired vector a~ a greater efficiency.
Once a comprehensive cDNA library is const~ucted from a cell line, desired genes can be identified with the assistance of subtractive hyblidization(see for example Sargent T.D., 1987~ A~eth. Enymol., Vol. 152, pp. 423-432;
Lee et al., 1991, P~oc~ N~l. Acad. SCi.J US~, Vol. 88, pp. 282~-2830). A
general method for subtractive hyb~id~tion is as ~ollows. The complementary s~and of the cDNA is synthesized and radiolabelled. This single st~nd of cDNA ~ be made from polyA mRNA or frDm the existing cDNA library. ~e radiolabelled cDNA is hyb~idized to a large excess of mRNA from a closely rehted cell population. After hybAdization the cDNA:mRNA hybrids are ~emoved from the solutioD by cbnnnatograp~ly on a hydsoxylapatite column. The emaining "subt~cted" radiolabelled cDNA can then be used to sc~eerl a cDNA
or genomic I)NA lib~aly of the same cell population.
~ ::
Subt~active hy~idi~ation removes the majority of the genes expressed in both cell popuht~ons and~thus en~iches for genes ~vhich are present only in the desired~ cell population. However, if the expression of a particular mRNA
se~uence is only a few times ~more abundant in the desired ~ell population than the subtractive population it may not be possible to isola~e the gene by subt~c1;ive hyb~idixation.
Summary of the InventioD
We have discovered a method for iden~i~ying, isola~ing and cloning mRNAs as cDNAs using a polymerase ampli~lcation method t~at employs at least two oligodeoxynucleotide primers.: In one approach, the first primer contains sequence eapable of hybridi~ing to a site including sequence that is immediatelyupst~am o~ the first A ribonuc1eotide of the mRNA's polyA tail and the second plimer corltains ar'oitra~ sequence.: In another approach, the first p~ner WO g3/18176 2 ~ ~ 2 ~ 8 ~ PCr/US93/022~

contains sequence capable of hybridizing to a site including the mRNA's polyA
signal sequence and the second prLme~ contains aIbit~y sequence. In another approach, the first primer contains a~ ary sequence and the second primer contains sequence capable of hybndi~ing to a site including the mRNA's Ko~ak S sequence. In another approach, the first p~imer contains a sequence that is substantiaLly eomplementaIy to ~he sequence of a mXNA having a known sequence and the second primer contains ar'oitraly sequence. In another approach, the ~lrst p~er contains aioitrary sequence and the second primer contains sequence that is substantially identical to the sequence of a mRNA
10 haviDg a l~own sesluence. The f~t primer is used as a primer for reverse transcrîption of the mRNA and the resultant cDNA is amplified with a polymerase using bo~h the f~t and second p~ers as a p~imer set.
Using this method with dif~erent pairs of the alterable p~rners, virtually any or all o~ the mRNAs ~rom any cell type or any stage of the cell cycle, 15 ~ including very low abundance mRNAs, can be identifled and isolated.
Additionally a comparison of the mRNAs from closely related cells, whieh may be f~ example at different stages of development or different stages of the cellcycle, can show which of the mRNAs are constitutively expressed and w}lich are differentially expressed, and their respective frequencies of expression.
The "filst pnmer" or ~t oligodeoxynucleotide" as used herein is de~med as ~ the oligodeoxynucleotide pIimer that is used for the reverse t~anscription of the ~RNA to make the first cDNA strand, and then is also used for amphfication of the cDNA. The~first p~ner can also be refer~ed to as the 3' pIimer, as this primer will ;hy~ridize ~o the mR~A and will def~ne the 3' end of~he first cDNA st~and. The "secQnd p~ner~ as used hereLn is de~med as being the oligodeoxynucle~tide primer that is used to ma~ce the second cDNA strand, and is also used for the amplification of the cDNA. Ihe second p~imer may also be referred to as ~he 5' primer, as ~his primer will hybIidize to the first cDNAstIand and will de~me the 5' end of the second cDN~ stIand.
The Uar'oitrary" sequence of an oligodeoxynucleotide primer as used herein ~: is defined as being b~sed upon or subject to individual judgement or diseretion.
In some instances, the arohraly sequence can be en~irely ~ndom or partly WO 93/18176 Pcr/us93/o2246 random for one or more bases. In other installces the arbitrary sequence can be selected to contain a specific ratio of each deoxynucleotide, for example approximately equal proportions of each deoxynucleotide or predominantly one deoxynucleotide, or to not contain a specific deoxynucleotide. The aioi~y S se~uence can be selected to contain, or not to contain, a recognition site forspecific restriction endonuclease. The a~ r sequence can be selected to either contain a sequence that is substantially identical (at least 50 homologous) to a mRNA of hlown sequence or to not contain sequence from a mRNA of known sequence.
An oligodeoxynuceotide primer can be either Ucomplementary" to a sequence or Usubstantially identical" to a sequence. As defimed herein, a complementaly oligodeoxynucleotide pnmer is a primer that contains a sequence which will hyb~ize to an mRNA, that is the bases are complementa~ to each other and a reverse transcriptase will be able to extend the primer to form a 15 cDNA strand of the :RNA. As defined here~, a substantially identical primer is~ a primer that contains~ sequence which is the same as the sequence of an m~NA, that is g~ r than S0~ identical, and the primer has the same orientation as an mRNA thus it will no~ hybridize to, or complement, an mRNA
but sùch~a primer can be used to hybddize to t~le fîrst cDNA st~and and can be 20 ~ e~tended~by a polyme~se to gener~te the second cDNA strand. The terms of art hybridization" ~or hybridize", as~used; herein, are defLned to be the base pairing of an oligodeoxynucleotide~pIimer with a mRNA or cDNA strand. The "conditions undér wl~ich~ an oligod~xynucieotide hyb~idizes with an mRNA or a cDNA, as used herein, 1s~defined to~ température and buffer conditioDs (that 25 are descAbed later) under whic}l the~ base painng of the oligodeoxynucleotide : ` ~
primer with either an~ rnRNA or a cDNA occurs and only a few mismatches (one or two) of the base painng;~are ~pe issible.
An oligohucleotide pIimer ~can contain a sequence that is hlown to be a consensus sequencen of an mRNA of hlown~ sequence. As defined herein, a 30 consensus sequence'7 is a ~sequence that has b~ found in a gene family of pr~oteins having a similar function or sill~ilar pr~perties. The use of a primer that : ~ ::

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WO 93/18176 PCr/US93/02246 ,.

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includes a consensus sequence may result in the clonis~g of additional members of a desired gene family.
The pre~e~ed length" of an oligodeoxynucleotide primer, as used herein, is determined from the desired SpeCi:~lCity of annealing and the number of S oligodeoxynucleotides having the desir~d specificity that are required to hybAdize to all the mRNAs in a cell. An oligodeoxynucleotide prim~r of 20 nucleotides is mo~e specific than an oligodeoxynucleotide primer of 10 nucleotides; however, add;tion of each random nucleotide to an oligodeoxynucleotide primer increases by four the number of oligodeoxynucleotide p~ers required in order to 10 l~ybridize to every mRNA in a cell.
In one aspec~, in geneIal, the invention features a method for identifying and isolating ~As by priming a preparation of mRNA for r~velse transcription with a first oligodeoxynucleotide primer that eontains sequence capable of hybridizing to a site including sequence that is immediately upstream15 of the ~'r~t A ribonucleotide of the mRNA's polyA tail, and amplifying the cDNA by a polyme~ase amplification method using t~e first primer and a second oligodeoxynucl~tide primer, for example a primer having arbit~y sequence, as : ~ a primer set.
In preferred embodiments, the first pIimer con~ains at least 1 nucleotide at 20 the 3' end of the olig~deoxymlcleotide that can hyb~idize to an mRNA sequencethat~ is immediately upstream of the polyA tail, and contains at least 11 ~ ~ : nucleotides $ the 5' end t~at will hybridize to the polyA tail. The entire 3' oligodeoxynucleodde is :preferably ~t least 13 nucleo~es ~n length, and can be up to 2Q llucleotides in lengt~.
: ~ : : 25 Most preferably, the first pnmer corltains 2 nucleotides at t}le 3' end of the oligodeoxynucleotide that can hybridi~e to an mRNA sequence that is ~mediately upst~am of the polyA tail. Prefe~ably~ the 2 polyA-non- -complementaly nucle~es are of the sequence 'VNj where ~ is deoxyadenylate ("dA"), deoxyguanylate (UdG), or deoxycytidylate ~dC"), and N, the 3' 30 tenninal nucleotide, is dA, dG, dC, or deoxythymidylate (UdT"). Thus the sequence o~ a pleferred first pnrner is s~-n lseq. ID. No. l].
~he use of 2 nucleotides can provide accurate positioning of the first plimer at wo 93/18176 ~ ¦ 0 2 7 8 4 Pcr/us93/o~246 the junction between ~he mRNA and its polyA tail, as the properly aligned oligodeoxynucleotide:mRN~ hybrids are more stable than improperly aligned hybrids, and thus the properly aligned hybrids will form and remain hybridized at higher temperatures. In preferred applications~ the mRNA sample will be S divided into at least twelve aliquots and one of the 12 poss;ble VN sequences of the fLrst pr~ner will be used ill each reaction to prime the reverse transcription of the mRNA. The use of an oligodeoxynucleotide with a single sequence will r~duce the number of mRNAs to be analyzed in eac}~ sample by binding to a subset of the mRNAs, statistically l/12th, thus simplifying the identi~lcation of 10 the m~As in each sample.
In some embodiments; the 3' end of the f~rst prirner ~ have 1 nucleotide that can hybridize to an ~A sequence that is immediately upstream of the polyA tail, and l2 nucleotides at t~e 5' end that will ~ybridize to the polyA tail, thus the p~ner will have the sequence S'-~nmT~ [Seq. ID. No. 2].
: ~ 15 The use of a sLngle non-polyA-eomplementaIy deoxynucleotide ~vol~ld decrease the number of oiigodeo~ynucleotides that are required to identify every mRNA to 3, however, the use of a~single nucleotide to position ~he annealing of primer ~o the jun~n of the mRNA ~uence and the polyA tail may ~esult in a Si~ifilCallt loss of specificity of the annealing and 2 non-polyA-complementary 20 ~ueleotides are prefe~.
~,~
In some embodimellts, the 3' elld of the first primer call have 3 or more nucleotides that can hybridize to an mRNA sequence that is immediately upstream o~ ~he polyA tail. l~e addition of each nucleotide to the 3' end wi~
~urther increase the stability ~ properly aligned hybrids, and the sequence to 25 hybridize to the polyA tail can be decreased by one nucleoLide for each additional non-l)olyA-complementary nucleotide added. The use of such a first primer may not be practical for rapid screening of the mRNAs con~ained within a given cell e, as the use of a ~Irst primer with more than 2 nucleotides that hybridize ~o ~: the mRN~ immediately upstream of the polyA tail significantly increases the 30 number of oligodeoxynucleotides required to identify every mRNA. For instanee, the pnmer 5' Tlll l ITI~ [Seq. ID. No. 3] would require the use of 48 separate f~st primers in order to bind to every mRNA, and would WO 93/1$176 ?.) 1 ~ 2 7 8 ~ PCI /US93/0224b significantiy inc~ease the number of reactions required to scr~n the mRNA from a given cell line. The use of oligodeoxynucleotides with a single random nucleotide in one position as a group of four can circumvent the problem of needing to set up 48 separate reactions in order to identify every mRNA.
S However as the non-polyA-complementary sequence became longer, it would quickly become necessary to increase the number of reactions re~quired to identify every mRNA.
In prefened embodiments, the second primer is of a~bit~ sequence and is at least 9 nucleotides in length. Preferably the second p~ner is at most 13 10 nucleotides in length and can be up to 20 nucleotides in length.
In anothe~ a~pect, ir gene~al, the invention features a method for p~epa~g and isola~g mRNAs by p~ing a pre~aration of mRNA for reverse t~anscAption with a fi~t pIimer that contains a sequence capable of hybridizing to the polyadeny~ation ~ignal sequence and at least 4 nucleotides that are 15 positioned 5', or 3', or both of the polyadenylation signal sequencç; this entire first pIimer is preferably at least 10 nucleotides in length, and can Ibe up to 20 nucleotides in length. In one prefe~ed embodiment the sequence S'-NNTTTAITNN [Seq. ID. No. 4] can be chosen such th~t t~e sequence is 5 '-GClTrAlTNC lSeq. ID. No. 53, and the ~our ~esul~ant p~ers are used 20 togethor in a single reaction for the plimi~ of the ml~NA for reverse transcription. Once the first cDNA st~and has been formed by reverse tl~scrip~on ~n the first primer can h uscd ~it}l a second primer, ~or example and aioi~y seguence primer, for the ampliflCatiOII of the cDNA.
In one aspect, in gene~al, the invention features a method for ident~ying 25 and isolating mRN~s: by primillg a :p~paration of mRNA for r~verse ttanscription~ with a fL~st oligodeox~mucleo~ide pnmer to generate a first cDNA
s~d, and p~in~ing the p:epar~tion of the second cDNA st~nd with a second primer that ~ontains sequence subs~antially identical to the Kozak sequence of mRNA, and amplifying the cDNA by a polymerase amplification method using 30 the first and second primers as a p~imer set.
In prefe~ed embodiments, the first and second primers are at least 9 deoxynucleo~ides in length, and are at most 13 nucleotides in length, and can be Wo 93/18176 21 0 ~ 7 8 4 Pcr/us93/o2246 up to 20 nucleotides in length. Most prefetably the first and second primers are10 deoxynucleotides in length.
In prefe~Ted embodiments the sequence of the first p~mer is selected at random, or the first primer eon~ains a selected arbitrary sequence, or the firstS primer co~tains a restriction endonuclease recogr~ition sequence.
In preferred em~ents the sequence of the second primer that contains sequence substantially identical to the Ko~k sequ~nce of mRNA has the sequence NNNANNATGN lSeq. ID No. 6}, or has the sequence NNNANN~TGG [Seq. ID No. 7~. Where N is any of the four 10 deoxynucleotides. P~eferably, the second pIimer has the sequence GCCACCATGG tSeq. ID No. g~. In some embodiments the first pIimer may further inelllde a restriction endonuclease recognition sequence that is added to either the 5' or 3' end of the plimer increasing the length of the primer by at least 5 nueleotides.
In another a~ct, in gene~al, the invention features a method for iden~ifying and isolati~g mRNAs by pnming a preparation of mRNA for r~verse anscrip~ion with a first oligodeoxynucleotide primer that ~ontah~s sequence thatis substan~ally complementaIy to the sequence of a mRN~ having a known ~: s~quence, and priming ~Ihe p~pal;ltion of the second cDNA st~nd with a second 20 ~ primer and, amplifying the cDNA by a polymerase amplification met~od USitlg the first and second pnm~s as a p~imer set.
In prerened embodiments, the ~irst and second pIimers are at least 9 deoxynucleotides in lellgth, and~are at most :13 tlucleotides in length, and can be up to 20 nucleotides in length. Mos~ pr~fe~ably the first and second p~mers are ~: : 2~ lO deoxynucleotides in length.~ ~:
In prefe~ embodiments the~sequence of the first primer fu~ther includes a rest~iction end~uclease sequence, which may be included within the prefelTed :
10 nu~leo~ides of the p~mer or may:be added to either the 3' or 5' end of ~he :
~; pIimer increasing the length of the oligodeoxynucleotide primer by at least S
: :: 30 nucleo~ides.

WO 93/18176 Pcr/vss3/o2246 ~lO~q-~?4 - 10-In prefer~d embodiments the sequence of the second pIimer is selected at random, or the second pnmer contains a selected arbit~y sequencel or the second primer contains a restriction endonuclease recognition sequerlce.
In another aspect, in general~ the invention features a method for S identifying and isolating mRNAs by p~iming a prepasation of mRNA for reverse ~nscription with a first oligodeo~rnucleotide primer, and p~ing the preparation of the second cDNA strand with a second pIimer that contaLns sequence ~hat is substantially identical to the sequence of a mRNA having a known sequence and, amplifyin~g the cDNA by a polymerase amplification 10 method using the first and second pnmers as a prirner set~
In p~eferred embodiments, the ~rst and secvnd primers are at least 9 de~xynucleotides in length, and are at most 13 nusleotides in length, and can beup to ~0 nucleotides in length. Most preferably the first and second pnmers are 10 deoxynucleotides in length.
In prefer~ed em~ents the sequence of the first primer is sele~ted at ~ndom, or the fLrst pnmer contains a selected aioit~uy sequence, or the first ~p~mer contains a restriction endonuclease re~gnition se~quence.
L~l prefened embodiments the sequence of the second pIimer having a sesluence that is substantially complementary to the sequence of all mRNA having20 a hlown sequen~e fur~her includes a restriction endonuclease sequence, which may be includ~ within the prefe~red 10 nucleotides of t~e primer or may be added to either the 3 ' or 5' end of the p~imer increasing the len~h of the oligodeoxynucleotide pr~ner by at least S mlcleotides.
In another a~pect, in geneIal, the invention features a method ~or 25 identifying and isolating mRNAs by priming a prepa~ation of mRNA ~or reverse t~nscription with a first oligodeoxynucleotide p~mer that conta;ns sequence thatis substant~ly complementary to the sequerlce of a mgNA having a hlown sequence, and p~ing the prepa~tion of the second cDNA strand with a second p~imer that contains sequence that is substantially identical ~o the Kozak seguence 30 of mRN~, and amplifying the cDNA by a polymerase amplification method using the first and s~cond primers as a primer set.

W~ 93/1~176 2 ~ Q 2 7 8 4 P~r~usg3/02246 In prefe~ed embodiments, the f~st and second primers are at least 9 deoxynucleotides in length, and are at most l3 nucleotides in length, and can beup to 20 nucleotides in length. Most preferably the first and second p~imers are10 deoxynucleotides in length.
S In snme preferred embodirnents of each of the general aspects of the invelltion, the amplified cDNAs are sepa~ated and then the desired cDNAs are reamplified using a polymerase amplification reaction and the first and second oligodeoxynucleotide primers.
In pr~ferred embodiments of each of the general aspects of the invention~ a 10 set of first and second oligodeoxynucleo~ide primers can be used, consisting of more than one of each primer. In some embodiments more than one of the first primer will be included in t}le reverse transc~iption naction and more th~ one each of the ~Irst and second p~ers will be included in the amplification reactions. The use of mo~e than orle of eac}l plimer will increase the number of15 m~As ide~ ed in each reaction, and the total nurnber of p~mers to be used will be dete~nined based upon the desir~dl method of sepaIating the eDNAs such ~at it remains possible to fillly isola~e each individual cDNA. In preferred em~ents a ~ew hur~dred cDNAs can be isolated and identified us~g denatu~g polyacrylamide gel electrophoresis.
Tbe method according to the invention is a significant advance over current clonilag techniques that utilize subt~ctive hybridization. In one aspect, the method according to the irlvention enables the genes which are ~Itered in the~
frequency of ~ression~ as well as of mRNAs which a~e constitutively and differer~ lly ~x~ressed, eo be identified by s~ple visual inspes~ion and isolated.
25 In another aspect the method accor~g to the invention prwides specific oligodeoxynucl¢otide p~imers for amplification of the desired mRNA as cDNA
and makes unneces~y an in~ermedia~g step of adding a homopolymeric ~ail to the f~t cDNA shand for priming of the second cDNA st~nd and thereby avoiding any interference ~rom the hornopolymeric tail with subsequent analysis 30 of the isolated gene and its product. In an~ther as~ect the m~thod according to the invention a~ows the cloning and sequenc~ng of selected mRNAs, so that the 21027~
Wo 93/18176 pcr/lJss3/o2246 investigator may determine the relative desi~bilit~y of the gene prior to screening a comprehensive cDNA libraly for the full length gene product.
Description of the Preferred limbodiments Drawin~s S Fig. 1 is a schematic representation of the method according to the invention.
Fig. 2 is the sequence of the 3' end of the Nl gene from normal mouse fibroblast cells (A31) [Seq. ID. No. 9~. ~
Fig. 3 is the Northern blot of the ~1 sequence on total cellular RNA from no~nal and tumongenic mouse fib}oblast cells.
Fig. 4 is a sequencing gel showing the results of ampliflcation ~or m~A
prepared from four sources ~lanes 1-4), using the Kozak primer alone, the AP-l primer alone, t~e Kozak and AP-l p~ners, the Kozak and AP-2 primers, the Kozak and AP-3 p~ners, the Kozk and AP-4 primers and tlhe Kozak and AP-5 pnmers. This gel will be m~r~ fully described later.
Fig. 5 is a partial sequence of tlhe 5' end of a clons, Kl, that was cloned f~om the Al-5 cell line ~hat was cultured at the rlon-peImissive tempe~ature andthen shi~ed to the permissive temperature (32.5C) for 24 h prior to the pr~ ation of the mRNA. The Al-5 cetl line is f~m a p~nary rat embryo fibroblast cell line that has been doulbly transfonned with ras and a eemp~rature sensitive mutation of P3 ~P31J~
Genelal Description!_Development of the Method By way of illustration a des~ription of examples ~ the method of the invendon follows, with a~description by way o~ guidance of how the particular illustIa~ve examples were de~elo~.
It is ~mpor~ for operation of the method that the length of ~he oligodeoxynucleotide be ~ppropria~e ~or SpeCiflC hybridization to rnRNA. In order to obtain specific hybridization, whet~er for conventional cloning methodsor PCR, sligodeoxynucleotides are usually chosen to be 20 or more nucleotides in length. The use af long oligodeoxynueleotides in this instance would dec~easethe number of m~NAs identified during each t~ial and would gleatly increase the number of oligodeoxynucleotides required to identify ~very mRNA. Recently, it WO 93/18176 2 1 0 2 7 8 4 Pcr/uss3/o2246 was demonstrated that 9-10 nucleotide primers can be used for DNA
polymoIp~lism analysis by PCR (Williams et aL, 1991, Nuc. Acids Res., Vol.
18, pp. 6531-6535).
The plasmid containing the cloned murine thymidine lcinase gene ("1~
S cDNA plasmid") was used as a model template to determine the required lengths of oligodeoxynucleotides for specific hybridization to a mRNA, and for the pr~duction of SpeCiflC PCR products. The oligodeoxylwcleotide pnmer chosen to hybridLze internally in the mRNA was varied between 6 and 13 nucleotides in length, and the oligodeoxynucleotide pIimer chosen to hybridize at the upstream 10 end of the polyA tail was varied between 7 and 14 nucleotides in lengtb. After nume~ous trials with dif~erent sets and lengths of primers, it was determined that the anne~g temperature of 42C is optimal for product specificity and the internally hybridizing oligodeoxynucleotide should be at least 9 nucleotides in length and a oligodeox~rnucle~tide that is at least 13 nucleotides in length is 15 requ~ to bind to the upstream end of the polyA tail.
With reference now to Fig. 1, the method according ~o the invention is depicted schematically. The mRNAs are mixed with the ~lrst primer, for e~ample lTnTrml~vN [Seq. ID. No. 2] ~T~VN) 1, and ~everse ~anscribed 2 to make the first cDNA strand. The cDNA is amplifiled as follows.
20 The first cDNA strand is added to the secQnd p~mer and the first primer and the polymerase in the standard buffer with the appr~priate concenl:rations of nucleotides and ~he componcnts are heated to 94C to denature the mRNA:cDMA
hybrid 3, the tempelature is reduced to 42~C ~o a11Ow the second primer to anneal 4, and then the temperature is incr~ased to 72C to allow the polymerase 25 to ex:tend the second p~mer S.: The cycling of t~le temperature is ~hen re~eated 6, 7, 8, to be~ the amplifica~ion of the sequences whieh are hybridized by the f3rst and second primers. The temperature is cycled until the desired number of copies of each sequence have been made.
As is well known in the art, this ampliflcation method ean be a~complished 30 using thennal stable polymerase or a polymerase that is not thermal stable.
When a polyme~se tha~ is not thermal stable is used, fresh polymerase must be added after the annealing of the pIimers to the templates a~ the start of the WO 93/18176 PCr/US93/0~246 elongation or extending step, and the extension step must be ~arried out at a temperature ~hat is pe~nissible for the chosen polymerase.
The following examples of the method of the invention are presented for illustrative pulposes only. As will be appreciated, the method according to the S invention can be used for the isolation of polyA mRNA from any source and can be used to isolate genes expressed either differentially or constitutively at any level, f~om rare to abulldant.
l~xample 1 ~perimentation with the conditions required for accurate and reproducible 10 results by PCR were conducted Wit~l the TK cDNA plasmid and a single set of oligodeoxynucle~tide primers; the sequence lTlTlT~CA (UT"CA") [Seq.
ID. No. 10] was chosen to hybridize to the upstream end of the polyA tail and the sequence CTIGAITGCC (ULtk3n) [Seq. ID. No. 11] was chosen to }lybridize 288 base pairs ("b~") ups~ of the polyA tail. The expected 15 fragment size using these two p~mers is 29g bp.
PCR was conducted under standard buf~er conditions well :hlown in the art : with lO ng TK cDNA plasmid (buffer and polymerase are available f~om Perkin Blmer-Cetus). ~e standard conditions we~e altered in that the primers were used at concentIations of 2.5 ~M T"CA ~Se~. ID. No. lOJ, 0.5 ~M L~3 [Seq.
20 ID. No. ll~, instead of 1 ,uM of each primer. The concentration of the nucleotides ("dNTPsn) was also varied ~ver a lO0 fold Iange, :~om the s~an~rd 200 ~M ~o 2 ,uM. ~e PCR pa~ameters were 40 cycles of a denaturing step for 30 seconds at 94C, an annealing st~p ~or 1 mirwte at 42C, and an extension step for 30 seconds at 72C. Significant amounts of non-~pecific PCR products 25 we~e observed when the dNI~ concent~tion was 200 ~M, concent~ations of dNTPs at or below 20 ~M yielded speeifically amplified PCR produc~s. The specificity of the PCR products was verified by restriction endonucleaæe digest of the amplified DNA, whic~ yielded the expected sizes of ~est~iction fragments. Insome instances it was ~ound that the use of up to S fold mo~e of the first primer 30 than the second prim~r also functioned to increase the sp~cifiGity of ~he product.
L~wenn~ the dNTP conGentration to 2 ~uM allowed the labelling of the PCR
products to a high specific activity with [c~-35S] dATP, 0.~5 ~M [a!-35S~ dATP ~Sp.

WCt 93J18176 2 :L O ~ 7 8 ~1 PCr/US93/02246 Act. 1200 Ci/mmol), which i~ necessary for distinguishing the PCR products when resolved by high resolution denaturing polyacrylamide gel electrophoresis, in this case a DNA sequencing gel.
13xampl~ 2 The PCR me~hod of amplification with short oligodeoxynucleotide plimers was then used to deteet a subse~ of mRNAs in mammaL;an cells. Total RNAs and mRNAs were pr~p~ from mouse ffbroblasts cells ~rhich were either grow~g rlormally, cyclingn, or serum sta~ved, Uquîescent". The RNAs and mRNAs were reverse ~scribed with T,ICA ~Seq. DD. No. IO] as the primer.
The T~CA p~mer [Seq. ID. No. 10] was annealed to the mRNA by heating ~he m~A and primer together to 65C and allowing the mix~ure to gradually cool to 35C. The r~verse tIanscription reaction was canied o~t with Moloney murine leukemia virus reverse t~ansc~i:p~ase at 35C. The resultant cDN~As were amplified by PCR in the presence of 'r1~CA [Seq. ID. No. 10] and 1~3 tSeq.
lS ID. No. 11], as des~ibed in ~ample 1, using 2 ,uM dNTPs. The use of the T~,CA ~Scq. ID. No. 10] and L~3 [Seq. ID. No. 111 primers allowed the TK
n~NA :t~ be used as an internal control for differential expression o~ a rare mRNA ~sc~ipt; TK mRNA is present at approximately 30 copies per cell.
~: Ihe DNA sequ~cing :gel revealed 50 to 100 amplifled mRNAs ~ the size range which is optimal for~ ther analysis, betw~n 100 to 500 nucleotides. The pat~ns of the mRNA speeies obse~ved in cycling and quiescent cells were véry sin~br as expec~ though some dif~erences were appa~ent. Notably, the 'rK
gene ~A, which is~exp~essed du~ng Gl and S :phase, was ~ound only in the RNA pl9~alatiODS from cycling cells, as expected, t~us demonst~ating the abiLityof this method to separate and isolate ra~ mRNA species.such as TK.
xample 3~
The expression of mRNAs in normal and tumorigenic mouse fibroblast :
;~ cells was also comp~ using: the TllCA [Seq. ID. No. 103 and I,tk3 ~Seq. ID.
~ No. 11] p~imers ~or the PCR~amplification. 'rhe mRNA was r~verse t~scrib~
30 using T~ICA [Seq. ID. No. 10] as the primer and the resultant cDNA was ~ ~ ampli~led by PCR`usLng 2 ~M dNTPs and the PCR parameters described above.
:~ The PCR products were sepaIated on a DNA sequencing gel. The 1~ mRNA

Wo 93/18176 Pcr/us93/o2246 was p~sent at the same level in both the normal and tumorigenic mRNA
pre~arations, as expected9 and provided a good intemal colltrol to demonstrate the re~resentation of rare mRNA species. Several other bands were present in one preparation and not in the other, with a few bands present in only the mRNA
S from norrnal cells and a ~ew bands present only in the mRNA from the tumorigenic cells; and some bands were expressed to different levels in the nonnal and tumorigenic cells. I~us9 the method according to the Lnvention can be used to identify genes which are norrnally continuously expressed (constitutive), and differentially expressed, suppressed, or otherwise altered 10 their level of expression.
Clonin~ of the m~A identifled in ~ample 3 Three cDNAs that ale, the TK cDNA, one cDNA expresse~ only in normal cells (Nl"), and one cDNA expressed only in tum~rigenic cells ("Tl"), were recover~d ~rom the DNA sequencing gel by electr~lution, ethanol 15 precipitated to remove ~he urea and other con~ninallts, and reamplified by PC~, in t~o eonsecutive PCR amplifications of 40 cycles each, with the primers TIICA
[Seq. II). No. 103 and ~ ~Seq. ID. No. 113 in the presence of 20 ~uM dNTPs to achieve optimal yield without compromising the ~pecificity. The reampli~
PCR products were co~ed to have the appropnate sizes and p~ner 20 dependencies as an additional control ~e reamplifled T~ cDNA was digested wlth two s~parate restriction endonucleases and the digestion proclucts were also conf~ed to be vf the co~Tect size.
The reamplified Nl [Seq. ~. No. 9J was cloned with the TA cloning system~ Invitrogen Inc., into the~pLasmid pCR1000 and sequenced. With 25 reference now to Fig. 2, the nu~leotide sequence clearly shows the Nl fragment tSeq. ID. No. 9] to be flanked by the unde~lined Ltk3 pIimer 15 at the 5' end and ~he underlined TI,CA primer 16 at the 3' end as expected.
A l?~orthern analysis of total cel~ular RNA using a radiolabelled Nl probe reconf~med ~at the Nl mRNA was only present in the normal mouse ~lbroblast 30 cells, and not iII the tumorigenic mouse fibroblast ~lls. With reference now to Fig. 3, the probe used to detect the mRNA is labelled to the right of the figure, and the size o~ the Nl mRNA can be estimated from the 28S and 18S markers wo 93/18176 2 1 0 ~ 7 8 4 Pcr/uss3/02246 di~picted ~o the left of the figure. The Nl mRNA is present at lo~ abundance in both exponentially growing and quiescent normal cells, lanes 1 and 3, and is absent from both exponentially growing or quiescent tumorigenic cells, l~nes 2 and 4. As a control, the same Northern blot was re~robed with a radiolabelled S probe for 36B4, a gene that is expressed in both normal and tumorigenic cells, to demons~te that equal amounts of mRNA, lanes 1-4, were present on the Northern blot.
E~ample 4 The cornparison of the expression of mRNAs in three cell lînes, one of w~ich was tested after culturing Imder two different conditions, was ~nducted.
The cell lines were a p~nary rat embryo fibroblast cell lille ("RE~F), the REF
cell line that has been doubly tIansformed with ras and a mutant of P3 ("T101-4"), and the R~F cell line that llas been doubly transformed with ras and a temperan~re sensitive mutation of P3 ("Al-5"). The Al-5 cell line was cultured ~: : 15 at the non-permissive temperature of 370C, and also cultured at 370C then shifted to the pe~missive temperature of 32.5oC ~or 24 h priorto the preparationofthe mRU~A. The me~hod o~heinve~on was conducted us~g ~he p ~ ers ~Ko ~ and on~ of five a~bi~uy sequence pruners, ~P-l, A~P-2, ~iP-3. ~iP-4, or A~P-5", a~the second and f~t p~ners~ ~espectively.
l~he s~quence of~the ~Kozak~ pr~ner was chosen based upon the pub~shed : consensus:~equence~forthe b~nsla~on s~ site consensus sequence of m~U~s' : ~Kozak, 1~911 Jou~ ~ell Bio~ogy, Vol. llS, pp. 887-903). A degenerate K~zak pr~ner h~v~g s~uences su~sban ia~y identical to the translation s~u~ site consensus se~ence wer~ used simultan~ously, thes¢ sequences we~e 5'-G~CRCCATGG [Seq. ID No. 12], in which the R is dA or dG and ~hus the oligodeoxy~ucleotide pIimer ~as only one of the given nucleotides which results in a mixture of pnmers.
The sequence of the five ar~ ary primers was a follows~ 1 had tbe sequence S'-AGCCAGCGAA ~Seq. II). No. 13]; AP-2 had the sequence 5'-GACCGCITGT ~Seq. ID. No. 14~; AP-3 had the sequence 5'-AGGTGACCCiT ~Seq. ID. No. 15]; AP~ had the sequence 5'-GGTACTCCAC
[Seq. ID. No. 1~; and AP-5 had the sequence 5!-Gl~GCGATCC tSeq. II). No.

6 Pcr/us93/o2246 17~. These arbit~ary sequence p~mers were chosen arbi~ily. In general each arbitrary sequence primer was chosen to have a GC content of 50-70%.
The mRNA was reverse t~anscribed using one of the AP primers, as the first primer, and the resultant first cDNA strand was amplifled in the presence of 5 both p~imers, the AP primer and the degenerate Kozak primer, by PCR using 2 ~M NTPs and the PCR pa~ameters described above. The PCR products were separated on a DNA sequencing gel. At least 50-100 amplified cDNA bands were present in each of the cell lines tested, and some bands were expressed to differ~nt levels in the different cell lines. As a control a reaction was conducted 10 using each arbitra~ primer in the absence of the Kozak p~ner. No cDNA was generated by the arbitra~y pI~mer alone, thus demonstr~ting that both primers were requi~ed to annp~fy an mRU~A in~D a cD N A.
With reference now to Fig. 4, the primer sets used for each reaction are shown at the top of the Fig. along the line marked Plimers. As a eontrol a .
15 reaction was conducted using the pIimers in the absence of mRNA, and using ~P-l with mRU~A in ~e absence ofthe Kozak pruner. No cDNA was generated by the p~ners ~nthe absence ofnnRU~A or by the albitrary pn~ner ~one, thus demonsbatLng that m~A is n~qu~ for amp~fication and that both p~uners were required to amplify an mRNA into a cDNA. The cDNA pr~ducts of the 20 ampliflcation w~re loaded in the same order ac~ss the gel, thus the R13F cell line is shown in each of lanes 1, cell l~ne T101-4 is shown in each of lanes 2, cel1 line Al-S cultured at 370C is shown in each of lanes 3, and CGll line Al-S
cultNred at 32.5o~ is shown ill each o~ lanes 4. ~ach pair of primers resulted in the:amplification of a different set of mRNAs from the cell lines. The reac~ions25 which were conducted using the Kozak p~mer and any of pnmers AP-l, AP-2, AP~, or AP-S as a pIimer set ~esulted in the ampliflcation of the same cDNA
patt~rn from each of ce11 lines REF, Tl01-4, Al-S cultured at 370C and Al-5 cultured at 32.5oC. The amplifleation of mRNA from each cell line and temperature using the: Kozak degenerate primer and the AP-3 primer resulted in 30 the fimding of one band in particu1ar which was present in the mRNA pr~pared from the Al-5 cell line when culbured at 32.5oC for 24 h, and not in any of the othe~ mRNA prepa~ations, as can be seen in Fig. 4 designated as Kl. Thus the WO 93~1~176 2 1 0 2 ~ 8 ~ Pcr/US93/02246 method according to the invention may be used to identify genes which are differentially expressed in mutant cell lines.
Clonin~ of the mRNA identified in ~xample 4 The cDNA (~KI'') that was expressed only in the Al-S cell line when S cultur~d at 32.5oC was recovered from the DNA sequencing gel and reampligledusing the primers Kozak and AP-3 as described above. The ~eampli~led Kl cDNA was confirmed to have the approp~iate size of approxLmately 450 bp, and was cloned with the TA clo~ing system, Invitrogen Inc., into the vector pCRII
(Invitrogen, Inc.) according to the manufacturers instructions, and sequenced.
With reference now to Fig. 5, the mlcleotide sequence clearl~ shows the Kl elone to be flanked by the underlined Kozak primer 20 at the 5 ' end and the underlined AP-3 p~mer 21 at the 3' end as expected. The S ' end of this partial cDNA is identified in Seq. D~ Nv. 18, and the 3' end of ~his cDNA is identifieclin Seq. ID No. l9. l~is partial sequence is an open reading ~rame, and a sea~ch of the gene databases EMBO and Genbank has revealed the ~nslated amino acid sequence from the 3' portion of Kl to be homologous to the ubiquitin conjugatingenzyme family (UB~ enzyme). Tbe t~nslated amino acid sequence of the 3' po;tion of Kl is 100% identical to a UBC enzyme from D. melanogaster a~d 75 % identical to the UBC-4 enzyme and 79 % identical to t}le UBC-5 en~yme ~om the yeast S. saccharo~ces; and 75 % identical to the UBC enzyme from Arabidopsis thaliana. I~e Kl c!oDe may contain the actual 5' end of this gene, otherwise the Kozak primer hybridized just after the 5 ' end. This result demonstIates th~t the met~od according to the invention can be used to clone theS' coding sequence of a gene Use The method according ~o the inve~tion can be used tv id~tify, isolate and clone mRNAs fr~m any number of sources. The method provides for the iden~lcation of des~e mRNAs by simple visual inspection ~er sepa~tion, and can be used for investigative research, industrial and medical applications.For instance, the reamplified eDNAs can be sequenced, or used to screen a DNA libraIy in order to obtain the full l~ngth g~ne. Once the se~uence of the cDNA is known, amino acid p~ptides can be made from the translatecl protein Wo ~3/18176 PC~/lJS93/02246 ~10Z781 20-sequence and used to raise antibodies. These antibodies can be used for further reisea~ch of the gene product and its function, or can be applied to medical diagnosis and prognosis. The reamplified cDNAs can be cloned into an approp~ate vector for fur~her propagation, or cloned into an appropriate S expression vector in order to be expressed, either in vitro or in vivo. The cDNAs whîch have been cloned into expression vectors can be used in industrial situations for oveIproduction of the protein product. In ot~er applications the reamplified cDNAs or their respective clones will be used as probes for in situ hybridization. Such probes can also be used for the diagnosis or prognosis of lQ disease.
Other Embodiments Other embodiments are within the following claims.
The length of the oligodeoxynucleotide can be va~ied dependent upon the ama~g tempe~ature chosen. In the preferred embodiments the temperature was chosen to be 42C and the oligonucleotide pr~ners were chosen to be at leas~ 9 llucleotides in len~th. If the annealing temperature were dec~eased to 35C then the oligonucleotide lengths can be decreased to at least 6 nucleotidesin length.
I~e cDNA could be radiolabelled with radioactive nucleotides other than 35S, such as 32p and 33P. When desi~, non-radioactive imagirlg methods can also ~e applied to the method ~cording to the invention.
The amplification of the cDNA could be accomplished by a tempera~ure cyc~g polyme~ase chain reaction, as was described, using a heat stable DNA
polyme~se for the rep~etitive copying of ~he cDNA while cycling the temperature for continuous rounds of denaturation, annealing and extension. Or the amplification could be accomplished by an isothennal DNA amplification method ~W~ker et al., 1992, Proc. Natl. Acad. Sci., Vo1. 89, pip. 392-396). The isothennal ampliflcation method would be adapted to use ior amplifying cDNA
by includi~g an appropriate restriction endonuclease sequence, one that will be nick~d at hemiphosphorothioate recognition sites and whose recognition site can be regenerated during synthesis with lX35S labelled dNTPs.

wo93~1~176 ~ 78 I p~r/vss3/o2~46 Proteins ha~ing similar function or similar functional domains are o~en referred to as bein~g par~ of a gene family. Many such pr~teLns have been clonedand identi~led to contain consensus sequences which are highly conserved ~non~st the members of the family. This conservation of sequence can be used S to design oligodeoxynucleotide pr~mers for the cloning of new members, or related members, of a family. Using the method of the invention ~he mRNA
from a cell can be reverse t~nscribed, and a cDNA could be amplified using at least one pnmer that has a sequence substantially identical to the sequence of amRNA of known sequence. Conser~sus sequences for at least the followin~g 10 families and functional domains l~ave been describ~l in the litelature: protein tyrosine kinases (Hanlcs et al., 1991, Met~ds on E~tzymolo~y, Vol. 200, pp. 38-81; Willcs, 1991, ~ethods in En~ymolog~y, Vol. 200, pp~ 533-546); homeobox ~enes; zinc-f~nger DNA binding proteins (Mi~er et al., 1985, E~BO Jo~lr., Vo1.
4, pp. 1609-1614); receptor proteins; the signal pept~de sequence of secr~ted 15 p~o~eills; proteins that localize to the ilucleus (Guiochon-Mantel et al., 1989, Vol. 57, pp. 1147-1154);~sese proteases; inhibitors of serine proteases;
~; c~o~cines; the SH2 and SH3 domains that have been described in tyrosine kinases and other pr~teins (Pawson et aL, 1992, Cell, Vol 71, pp. 359-362);
serine~ onine and ~rosine phosphatases (Cohen, 1991, Method~ in ~20: ~ En~ology, VoI. 201, pp. 398-408); cyclins and cyclin-dependent protein .
~: ~ kinases ~CDKs) (seefor ex., ~eyomarsi et al., 1993, Proc. Natl. Acad. Sci., U~, Vol. 90, pp. 1112-1116).
Primers for any consensus sequence ean ~eadily be designed ~ased upon the ;codon usage o~ the amino acids. I~e inco~poration of degeneracy at one or more 25 ~ sites allows the designillg of a primer which will hybridize to a high percentage, greater than 5Q%, of the mRNAs containing the deshed consensus sequence.
Primers for use in ~the method according to the invention could be designed based UpOD the consensus sequence of the zinc ~mger DNA binding proteins, ~or :;: example, based UpO21 the amino acid consensus sequence of the prot~ins PYVC.
30 IJseful plimers for the cloning of further members of this ~amily can have the ~ollowing sequences: 5'-GTAYGCNrGT [Seq. ID. No. 20~ or 5'-GTAYGCNTGC ~Seq. ID. No. 2lj, in which the Y refers to the WO 93~18176 Pcr/US93/02~4S
2t~278 ~ - 22 -deoxynucleotides dT or dC for which the p~mer is degenerate at this position, and the N refers to inosine ("I). The base inosine can pair with all of the other ~ases, and was chosen ~or this position of the oligodeoxynucleotide as thecodon for valine "V~ is highly degenerate in this position. The described 5 oligodeoxynucleotide primers as used will be a mixture of 5'-G'rATGCITGT
and 5'-GTACGCITGT or a mixture of 5'-GTATGCITGC and S '-GTACGCITGC .

,.-.

wo 93/lg176 ~ 1 0 2 7 8 ~ P~/US~3/~2246 SEQUENCE LISTING
(1) GEN~RAL INFOR~ATION:
(i~ APP~ICANT: Liang, Peng Pardee, Arthur B.
S (ii) TITLE OF I~VE~TION: IdeMtifying, Isolating and Cloning Mes~enger RNA~
(iii) NWMBER ~F SEQUENCES: 21 (iv) CORRESPONDE~CE ADDRESS:
(A) ADDRESSEE: Choate, Hall & Stewart 0 (B) STREET: Exchange Place, 53 State Street (C) CITY: Boston (D) STATE: Na~sachu~ett~
(E) COUNTRY: ~.S.A. :~
(F) æ~P: 02190 ~v) COMP~TER READAB~E FORM:
(A) MEDIUM TYPB: Floppy disk (B) coNæurER: IB~ PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARR: Patent~n Relea~e #1.0, Version ~1.25 (vi ) CU~RE~T APPLI~A~IO~ DATA:
(A) APPLICATION NUMBER: US
~B) FILIMG D~T~: ll-MAR-1993 ~ (C) CLASSIFICATION:
: : . (vii) PRIOR APPLIC~TION DAT~:
(A) APP~ICATION ~UMEER: US 07/850,343 (B) F$LI~G DATE: 11-MAR-1992 (viii) ~ Y~AGE~T INFORM~TION:
tA) NAME: Paster~ack, Sam (B) REGISTRATION N~NBER: 29,576 (C) R~FERENCE/DOCKET NV~BER: DFCI234CIP
(ix) TELECO~MUNICATION INFO~M~TIO~:
: (A) T~LEP~O~E;: 617:227-5020 (B) TELEFAX: 617 227-7566 (2) INFORMATION FOR SEQ ID ~0:1:
(i) SEQ~ENOE CHARAC~ERISTI~S:
(~) LE~GTH: 13 ba~e pair~
(B) TYPE~ nucleic acid (C) STRA~DEDWESS: single (D) TOPOLOGY: linear : 40 (ii) NDLECULE TYPE: other~nucleic acid tiii) HYPOTHETICAL: ~0 (iv):ANTI-SE~SE: NO
~xi) SEQU~CE DESCRIPTION: SEQ ID NO:l:
TT~mT TV~I ~ 13 (2) INFORM~TION FOR SEQ ID NO:2:

WO 93/18176 PC~US93/02246 )2~Rq~ - 24 -( i ) SEQUE~CE CR~RACTERISTICS:
(A) I,ENGTH: 13 base pair6 (E5) TXPE: nucleic acid (C) STR~NDEDNES5: ~ingle S tD) TOPOLOGY: linear (ii) MOLECULE TYPE: o~her nucleic acid (iii) HYPOTHETICAL: NO
(i~) ANTI-S~NSE: NO
(xi) SEQUE~CE DESCRIPTION: SEQ ID ~0:2:
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(i) SEQVENCE CHARACT~RISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear ~ii) ~OL~CULE TYPE: other nucleic acid (iii) HYPOTHETI~AL: NO
(iv) A~TI-SENSE: NO
(xi ) SEQUE~C~ DESCRIPTION: SEQ ID N~:3:
~3 (2) I~FORMATION FOR 5EQ ID NO:4:
(i) SEQUBNCE CHARAC~ERISTICS:
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~2) I~FORN~TIO~ FOR SEQ ID NO:5:
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(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CH~RACTERISTICS:

W O 93/1B176 ~ I 0 2 7 8 4 PCT/US93/02246 ~5 _ ~A) LENGTH: l0 ba~e pair~
(B) ~YPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOl~E~ICAL: NO
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(xi) SEQUENCE DESCRIPTION: 5EQ ID NO:6:
NN~AN~ATGN 10 (2) I~FORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHAR~CTERISTICS:
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(B) TYPE: nucleic acid (C) STRANDEDNESS: Bingle ~D) TOPOLOGY: linear (ii~ MOLECUL~ TYPE: other nucleic acid (iii) RYPO~HETICAL: NO
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~xi) S~Q~ENCE DESCRIPTION: 5~Q ID NO:7:
20 ~ TGG l0 (2) I~FORMATION POR SEQ ID NO:8:
(i) SBQU~NC~ CNhRPCTERISTICS:
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:~ :30 (xi) SEQUE~CE D~SCRIPTION SEQ ID NO:8:
G~CACCATGG l0 (2~ I~FORM~TIOW FOR S~Q ID NO:9:
(i) sEQ~ENcB:c~ARrcTERIsTIcs:
~: (A~: L~NGIH:: 260 ba~e pair~
~B) TYPE: nucleic acid (C) STRANDEDNESS: 6ingle (D) TOPO~OGY: linear tii) MOL~C~1E TYPE: cDNA
: (iii~ HYPOTHETICAL: NO
(iv) ~NTI-S~S~:~ NO
(xi) SEQU~NCE DESCRIPTION~: SEQ ID NO:9:
CTTG~TTGCC TCCTAC~GC~ GTTGCAGGCA CCTTTAGCTG TACCATGAAG TTC~CAGTCC 60 GGG~TTGTGA CCCTAATACT~GGAGTTCCAG ATGAAG~TGG ATATGATGAT GAATATGTGC l20 TGGAAGATCT TGA~GTA~CT GTGTCTGATC ATATTCAGAA GATACTAAAA CCTAACTTCG 180 CTGCTGCCTG GG~AGAGGTG GGAGGAGCAG CTGCGACAGA GCGTCCTCTT CACA~AGGGG 240 .

WO 93/lX176 PClr/US93/02246 210278~ 26-TCCTGG~TGA AArAAA~AAA 260 (2) INFOP~ TION FOR SEQ ID NO:10:
(i) SEQUE~CE C~ARACTERISTICS:
(A) LENGTH: 13 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYP~: other nucleic acid (iii) HYPOTHETICAL: NO
0 (iv) ANTI-SRNSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

~2) I~FORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) hENGT~I: 10 ba~e pair6 (B) TYPE: nucleic acid (C) STRA~DEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: other nucleic acid (iii) HYPOTHETICAL: ~0 (iv) A~I-SE~SE: MO
(xi) SEQUE~CE DESC~IPTION: SEQ ID NO:ll:

(2~ I~FORMATION FOR SEQ ID NO:12:
(i) S~QUE~CE CHARACTERISTICS:
(~ LENGTH: 10 ba6e ~air6 (B) TYPE: nucleic acid - (C) STRANDED~SS: ~ingle(D) TOPOLOGY: linear lii) ~OLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: ~0 (~) ANTI-S~SE: NO
(xi) SEQ~ENCE DESCRIPTION: SEQ ID ~0:12:

35 (2) INFORMATI~N FOR SEQ ID NO:13: :
(i) SEQU~CE CHARACTERISTICS:
(A) LENG~H: 10 base pairs (B) TYPE: nucleic acid (C) STRA~DEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE ~YPE: other nucleic acid (iii) HYPOT~ETIC~L: NO
~ i~) A~TI-SENSE: ~Q
~ xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

WO 93/18176 2 1 Q 2 7 8 ~ PCI/US93/0~46 (2~ INFO~MATION FOR SEQ ID NO:14:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGT~: 10 base pair~
~B) TYPE: nucleic acid S ~C) STR~NDEDNESS- ~ingle (D) TOPOLOGY: linear (ii~ NOLECUL~ TYPE: other nucleic acid (iii) HYPOTHETICAL NO
(iv) ~TI-SENSE: NO
0 (xi) SEQUE~CE DESCRIPTION: SEQ ~D NO:14:

(2) I~FORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LE~GTH: 10 base pairs (B) TYPE: nucleic acid ~C) STRAND~DWESS: ~ingle (D) TOPOLOGY: linear tii) MOLECULE TYPE: other nucleic acid ~iii) HYPOTHETICAL: NO
: 20 ( iv~ ~TI-5~NSE: NO
(xi) SEQUE~OE DESCRIPTION: SEQ ID NO:15:
AGGTGA~CGT 10 (2~ INFORM~TION FOR SEQ ID NO:16:
(i) SEQUE~CE CHA~CTERISTICS:
(A) LENGTH: 10 base pair~
(B) TYPE: nucleic a~id (C) STRANDED~SS: ~ingle (D) TOPOLOGY: linear ( ii ) ~0~ TYPE: o~her ~ucleic acid (iii) ~YPOTHETICAL: ~0 (iv) ANTI-SE~SE: NO
(xi) SEQ~E~E DESCRIPTION: SEQ ID NO:16:
GGTACTCCAC : 10 (2~ IN~ORMATYON FOR SEQ ID ~0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LE~GTH: 10 base pair6 (B~ TYPE: nucleic acid ~C) STRAN~EDNESS: ~ingle (D) TOPOLOG~: linear (ii) MO~ECULE TYPE: other nucleic acid (iii) H~POTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUE~CE DESCRIPTIO~: SEQ ID NO:17:

(2) I~FORNATION FOR SEQ ID NO:18:

2~ J7 8~ 28 (i) SEQUENCE C~ARACTERISTICS:
(A) LEMGI~1: 42 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Bingle S (D) TOPOLCGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: ~O
~iv) ANTI-SE~SE: NO
(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO:l8:
10 GCCGCCATGG CTCTGA~GAG AATCCACAAG GACACCCATG AA 42 (2) INFORMATIO~ FOR S~Q ID NO:l9:
(i) SEQUENC~ C~AR~CTERISTICS:
(A) LE~GTH: 78 ba~e pair~
~B) TYPE: ~ucleic acid (C) STR~DED~SS: single (D) TOPOLOGY: linea~
~ii) NOLEC~LE TYPE: cDNA
(iii) HYPOTHETI~AL: ~O
~iv) ANTI-SE~SE: ~O
(xi ) SEQUE~CE DE5CRIPTIO~: SEQ ID NO:l9:
~TTGCATTTA CA~CAAG~AT TTATCATCC~ A~TATTAACA GTA~TGGCAG CATTTGTCTT 6Q
G~TATTCTAC GGTC~CCT 78 (2) I~FORMATIO~ FOR SEQ ID ~0:20:
(i) 5EQ~ENC~ CHAR~CTERISTICS:
1~) ~E~GTH: 10 ba~e pairs : (~) TYPE: ~ucleic acid (C) STRAND~DNESS: ~ingle (D) TOPOIOGY- linear (ii) MOL~CUL~ TYPB: other nucleic acid (iii) HYPO~H~TICAL: ~0 (iv) ~TI-SENSE: ~0 (xi) SEQUENC~ DESCRIPTION: SEQ ID NO:20:
GTAYGC~TG~ ~ lO
(2) INFORMATION FOR 5EQ ID NO:21:
(i~ SEQ~E~CE CHARACTERISTICS:
(A) LENGTH: lO ba~ej palrB
(B) TYPE- nucleic acid (C) STRANDEDNESS: sinyle (D) TOPOLOGY: linear (ii) ~O~EC~LE TYPE: other nucleic acid (iii) HYP~THETICAL: NO
(i~) ANTI-SE~SE: NO
(xi) SEQ~ENCE D~SC~IPTION SÆQ lD ~0:21:
GTAYGC~TGC 10

Claims (36)

Claims

1. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising:
contacting the mRNA with a first oligonucleotide primer having a base sequence that is substantially common to all mRNAs, under conditions in which said first primer hybridizes with the mRNA at a site having said common base sequence, reverse transcribing the mRNA using a reverse transcriptase and said first primer to produce a first DNA strand complementary to at least a portion of the mRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes with the DNA strand at a site, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstreams from said site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a polymerase, and said first and second primers to clone the DNA.

2. The method of claim: 1 wherein said first primer hybridizes with the mRNA at a site that includes a polyA signal sequence.

3. The method of claim l wherein said first primer hybridizes to a portion of the polyadenosine (polyA) tail of said mRNA and at least one non-polyA
nucleotide immediately upstream of said portion.

4. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising:
contacting the mRNA with a first oligodeoxynucleotide primer under conditions in which said first primer hybridizes with mRNA at a site, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of themRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer having a base sequence that is substantially common to all mRNAs, under conditions in which said second primer hybridizes with the first DNA strand a site containing a complement of said substantially common base sequence, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from said site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers to clone the DNA.

5. The method of claim 4 wherein said second primer hybridizes with the first DNA strand at a site that includes a Kozak sequence.

6. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the mRNA with a first oligodeoxynucleotide primer under conditions in which said first primer hybridizes with mRNA at a site including asequence immediately upstream of a first A ribonucleotide of the mRNA's polyA
reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of themRNA upstream from the site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes which DNA, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from the site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers to clone the DNA.

7. The method of claims 6 wherein said first primer hybridizes with the mRNA at a site that includes at least one nucleotides upstream from and adjacentto the first A ribonucleotide of the polyA tail.

8. The method of claim 7 wherein said first primer hybridizes with the mRNA at a site that includes at least two nucleotides upstream from and adjacentto the first A ribonucleotide of the polyA tail.

9. The method of claim 6 wherein said first primer includes a polyA-complementary region comprising at least 11 nucleotides and, upstream from said polyA-complementary region a non-poly-A complementary region comprising at least one nucleotide.

10. The method of claim 9 wherein said non-polyA-complementary region comprises at least 2 contiguous nucleotides.

11. The method of claim 10 wherein said non-polyA-complementary region comprises 3'-NV, wherein V is one of deoxyadenosine, deoxycytidine, or deoxyguanosine, and N is one of deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine.

12. The method of claim 9 wherein said first primer comprises at least 13 nucleotides.

13. A non-specific cloning method for isolating m a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the with a first oligodeoxynucleotide primer under conditions in which said first primer hybridizes with mRNA at a site that includes the mRNA's polyA signal sequence, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first .ang. strand complementary to at least a portion of the mRNA upstream from the site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes with DNA, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from the site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers.

14. The method of claim 13 wherein said first primer comprises at least 6 deoxyribonucleotides.

15. The method of claim 13 wherein said first primer comprises at least 9 deoxyribonucleotides.

16. The method of claim 6 or 13 wherein said second primer comprises at least 6 deoxyribonucleotides.

17. The method of claim 6 or 13 wherein said second primer comprises at least 9 deoxyribonucleotides.

18. The method of claim 6 or 13 wherein said second primer includes a randomly selected nucleotide sequence.

19. The method of claim 6 or 13 wherein said first or second primer includes a selected arbitrary sequence.

20. The method of claim 6 or 13 wherein said first or the second primer includes deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.

21. The method of claim 6 or 13 wherein said first or second primer includes a restriction endonuclease recognition sequence.

22. The method of claim 6 or 13 wherein said second primer includes a sequence identical to a sequence contained within a mRNA of known sequence.

23. The method of claim 6 or 13 wherein at least one of said first or second primers comprises a plurality of oligodeoxynucleotides.

24. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the mRNA with a first oligodepxynucleotide primer under conditions in which said first primer hybridizes with mRNA at a site, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of themRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes with the DNA strand at a site, said site including a Kozak sequence, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from the site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers.

25. The method of claim 24 wherein said first oligodeoxynucleotide includes a sequence substantially identical to a sequence contained within an mRNA of known sequence.

26. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the mRNA with a first oligodeoxynucleotide primer, having a base sequence substantially complementary to a sequence in a mRNA of known sequence, under conditions in which said first primer hybridizes with mRNA at a site having said substantially identical sequence, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of themRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes with the DNA strand at a site, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from said site of hybridization of said primer with said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers.

27. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the mRNA with a first oligodeoxynucleotide primer under conditions in which said first primer hybridizes with mRNA at a site, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of the mRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer, having a sequence substantially identical to a sequence in a mRNA of known sequence, under conditions in which said second primer hybridizes with the first DNA strand at a site containing a complement of said substantially identical sequence, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from said site of hybridization of said second primer with said first DNA strand, and amplifying the first and second DNA strands using a polymerase and said first and second primers.

28. A non-specific cloning method for isolating in a nucleic acid sample a DNA complementary to a mRNA, comprising contacting the mRNA with a first oligodeoxynucleotide primer, having a sequence substantially complementary to a sequence in a mRNA of known sequence, under conditions in which said first primer hybridizes with mRNA at a site containing said substantially identical sequence, reverse transcribing the mRNA using a reverse transcriptase and said first primer, to produce a first DNA strand complementary to at least a portion of themRNA upstream from said site of hybridization of said first primer with the mRNA, contacting the first DNA strand with a second oligodeoxynucleotide primer under conditions in which said second primer hybridizes with the DNA strand at a site, said site including a Kozak sequence, extending the second primer using a DNA polymerase to produce a second DNA strand complementary to the first DNA strand downstream from said site of hybridization of said second primer with; said first DNA strand, and amplifying the first and second DNA strands using a DNA polymerase and said first and second primers.

29. The method of claim 24, 26, 27, or 28 wherein said first primer comprises at least 9 deoxyribonucleotides.

30. The method of claim 24, 26, 27, or 28 wherein said first primer comprises 10 deoxyribonucleotides.

31. The method of claim 24, 26, 27, or 28 wherein said second primer comprises at least 9 deoxyribonucleotides.

32. The method of claim 24, 26, 27, or 28 wherein said second primer comprises 10 deoxyribonucleotides.

33. The method of claim 24, 26, or 27 wherein said first primer is composed of a randomly selected sequence of deoxyribonucleotides.

34. The method of claim 24, 26, 27, or 28 wherein said first primer includes a selected arbitary sequence of deoxyribonucleotides.

35. The method of claim 24, 26, 27, or 28 wherein said first primer or said second primer includes a restriction endonuclease recognition sequence.

36. The method of claim 24, 26, 27, or 28 wherein at least one of said first or second primers comprises a plurality of oligodeoxynucleotides.