WO2002050310A9 - Rolling circle amplification detection of rna and dna - Google Patents
Rolling circle amplification detection of rna and dnaInfo
- Publication number
- WO2002050310A9 WO2002050310A9 PCT/US2001/049780 US0149780W WO0250310A9 WO 2002050310 A9 WO2002050310 A9 WO 2002050310A9 US 0149780 W US0149780 W US 0149780W WO 0250310 A9 WO0250310 A9 WO 0250310A9
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- dna
- cells
- exonuclease
- rolling circle
- circle amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- This invention relates to the detection of DNA and RNA.
- this invention relates to the detection of DNA and mRNA expression levels using rolling circle amplification.
- Rolling circle amplification is a nucleic amplification technique used with a 'padlock' oligonucleotide probe to detect single base changes in isolated nucleic acids (1-5).
- RCA is a powerful technique in theory, in practice it suffers from sensitivity and reproducibility problems.
- the present invention is directed to improved RCA techniques for the detection of DNA and RNA.
- the present invention is directed to improved RCA in situ techniques for the detection of DNA and RNA.
- the present invention is directed to the finding that in addition to restriction enzyme digestion of DNA, additional steps are required to achieve consistent and satisfactory results for RCA detection of DNA, particularly in situ DNA detection. Whereas heat denaturation is typically used to render the target DNA single stranded, the present invention is directed to the finding that complete removal of the non-targeted DNA strand by digestion with exonuclease significantly increases the efficiency of the RCA DNA detection process both in solution and in situ.
- the present invention is also directed to the use of RCA to detect mRNA in situ. Using appropriate image analysis techniques, the RCA assay is sufficiently quantitative to enable transcriptionally-mediated dose- response curves to be generated.
- Rolling circle amplification is a versatile technology used to locate single-base substitutions in DNA and RNA which has proven to be very useful in detecting point mutations in extracellular nucleic acid but has not as yet been successful at detecting base changes in situ.
- This invention is also directed to a method of employing RCA in situ to detect gene copy num er, single ase mutat ons an gene expression levels in individual cultured cells and in tissue sections. By pre-treating DNA with a restriction enzyme and an exonuclease, one strand of the DNA helix is removed. This produces a single-stranded nucleic acid template in which minimal DNA- induced stearic hindrance exists to inhibit the polymerase activity necessary for RCA detection of DNA.
- the present invention is directed to the use of RCA to detect simultaneously single base changes in genomic DNA and levels of gene expression by amplifying transcribed RNA as well as DNA.
- the present invention can be used to detect and track mutations in cancer sections, enabling the course of genetic progression to be studied in situ.
- the present invention is directed to a method of rolling circle amplification of DNA by: a) providing DNA; b) digesting the DNA with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to prepare the DNA for rolling circle amplification and d) performing rolling circle amplification on the DNA. Such methods can be performed in solution and in situ.
- the present invention is further directed to a method of preparing DNA for rolling circle amplification in situ, by a) fixing cells on a surface wherein the cells include DNA; b) digesting the DNA on the surface with an endonuclease to form nicked DNA; and c) digesting the nicked DNA with an exonuclease to prepare the DNA for rolling circle amplification.
- the invention is directed to a method of performing rolling circle amplification in situ, by a) fixing cells on a surface wherein the cells include DNA; b) digesting the DNA on the surface with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to form target DNA; d) ligating a padlock oligonucleotide probe to the target DNA to form ligated DNA and d) performing rolling circle amplification in situ on the ligated DNA.
- the present invention is directed to a method of performing rolling circle amplification in situ, including: a) providing cells embedded in paraffin wherein the cells contain DNA; b) digesting the DNA in the paraffin with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to form target DNA; d) ligating a padlock o gonuc eo e o t e arget o orm gate ; an e per orm ng rolling circle amplification in situ on the ligated DNA.
- the endonuclease is a restriction endonuclease and. Restriction endonucleases and exonuclease are commercially available from companies such as Promega Corporation,
- the surface is a microscope slide coverslip or a microscope slide.
- the cells utilized in the method of the invention may be prokaryotic, eukaryotic plants or fungi.
- the eukaryotic cells may be mammalian including human, reptile, amphibian, avian or plant cells.
- the prokaryotic cells may be bacterial cells.
- the DNA may be selected from eukaryotic, prokaryotic, viral, chromomosomal, mitochondrial or chloroplast
- the present invention is directed to a kit for rolling circle amplification, including: a) an exonuclease and b) an endonuclease.
- the kit may further include RCA reaction buffer.
- the kit may yet further include an oligonucleotide for RCA.
- the present invention is further directed to a method of detecting RNA in situ, including: a) fixing cells on a surface wherein the cells include RNA; performing rolling circle amplification in situ on the RNA to detect the RNA in situ.
- the surface is a microscope slide.
- the cells are centrifuged onto the microscope slide
- the cells are fixed by treatment with alcohol.
- the preferred alcohol is ethanol.
- the invention is directed to a method for performing rolling circle amplification in situ to detect RNA, including: a) centrifuging cells onto a surface; b) fixing the cells on the surface with ethanol; c) hybridizing a padlock oligonucleotide probe to the RNA to form a
- FIGURE 1 shows a schematic diagram of rolling circle amplification (RCA) in situ for detection of DNA.
- FIGURE 2 shows the use of RCA in Human Lymphoblastoid
- HLB cells in the presence of digoxigenin-dUTP(A) or biotin-dUTP(B) for copy number detection.
- FIGURE 3 shows a Molt-4 cell in which a single nucleotide (G to A) difference in two alleles of the Tp53 gene was detected by RCA in situ.
- FIGURE 4 shows mRNA detection in normal HLB cells(A) and Molt-4 cells(B) stained with acridine orange.
- FIGURE 5 shows radiation dose response curves of HLB cells.
- Rolling Circle Amplification is a molecular cytogenetic technique used with a 'padlock' oligonucleotide probe to detect single base changes in isolated nucleic acids. At 10 bases per helical turn, the hybridized probe wraps around its target 3 times, and the remaining 70 bases form an unhybridized single- stranded loop. Post-hybridization DNA ligation connects the two ends of the probe in the middle of the 30 base binding region. The unbound 70 base loop facilitates probe circularization and permits approximately 20 bases to serve as a primer recognition site for DNA polymerase to replicate the circle. Rolling circle amplification may be performed in solution or in situ.
- PCR Polymerase Chain Reaction
- PCR The polymerase chain reaction (PCR) is a technique utilized to amplify DNA. Typical PCR reactions include appropriate PCR buffers, DNA polymerase and one or more oligonucleotide primers. Various modifications of PCR techniques are possible as detailed in Current Protocols in Molecular Biology ed. F.M. Ausubel, R. Brent, D.D. Moore, K. Struhle, Massachusetts General Hospital an arvar e ca c oo (1987 w ch is ereby incorporated by reference.
- Oligonucleotide primers are short chains of nucleotides useful in RCA technigues, PCR techniques,
- DNA or RNA either in solution or in situ.
- Padlock probes are probes comprised of approximately 100 nucleotides which hybridize to targets of approximately 30 bases and find use in RCA.
- the 30-base target-binding region of the probe is split into two approximately 15-base segments placed in opposite orientation at each end of the linear probe so that a circle must be formed for hybridization to occur (6, 7).
- RCA in situ is similar to conventional RCA in solution except that the RCA reaction mix is added to a chromosome template on a surface such as a microscope slide or cover slip or to a formalin-fixed paraffin-embedded tissue section rather than in a microfuge tube.
- the RCA products remain closely associated with their target sequences.
- the reaction products can be identified, if necessary, by incorporating a dNTP-conjugated fluorochrome to the RCA solution.
- Endonuclease An endonuclease is an enzyme that makes a nick in both strands of double stranded DNA. Such endonucleases are available commerecially from manufacturers such as Promega Corporation,
- Exonuclease An exonuclease is an enzyme that digests double stranded DNA from a 3' end or a 5' end leaving a single strand of DNA in its wake.
- Exemplary but not limiting examples of exonucleases which find use in the invention include 5' exonucleases, 3' exonucleases including Exonuclease
- Rolling C rcle Amp ification is a molecular cytogenetic. technique used in conjunction with a 'padlock' oligonucleotide probe to locate and detect single base changes in DNA and RNA. The padlock probe is used to locate the individual base, and RCA is used to create a large enough signal to be detectable by conventional microscopy.
- Oligonucleotides are short linear strands of synthetic
- DNA that are frequently used as probes can be hybridized to DNA or RNA, either in solution or in situ, and are generally detected by fluorescence.
- the fluorescing of the oligos is achieved by attaching either a fluorescent molecule or an antigen (to which a fluorescently tagged antibody is later attached) directly to the oligo. This works well when the probe is thousands of bases long, but as the length of the probe decreases, the amount of fluorescent label that can be attached to the probe also decreases. This generally limits the length of probe that can be detected to approximately 1000 bases.
- Padlock probes were designed to lower this limit of detection
- Padlock probes are oligos about 100 bases long, designed to hybridize to a segment of nucleic acid less than half that length, perhaps 30 bases. Of those 30 bases, approximately 15 are represented on one end of the padlock probe and approximately 15 on the other end. Thus, when the padlock probe hybridizes to its target, the probe forms a circle. Due to the fact that DNA is a helix, and makes a full turn every 10 bases, in 30 bases the probe is wrapped around its DNA target 3 times, with the remaining 70 bases forming the unhybridized part of the loop. Post-hybridization treatment with a DNA ligase attaches the two ends of the oligo to one another, in the middle of the 30 base binding segment.
- the 70 base segment of the loop that is not bound to the DNA target may be 'junk' sequence, serving to facilitate circularizing the probe and facilitating primer recognition.
- a DNA primer a small oligo such as used to provide the starting point for a PCR reaction
- An isothermal process the polymerase progresses cont nuous y aroun t e oop unt t e 0 ase circle has been replicated hundreds or thousands of times.
- the padlock probe can be designed to circularize in such a way as to place the gap between the two end bases directly over a particular base on a target. If one wants to determine whether a particular base in a gene has been changed from, for example, guanine to adenosine, one simply makes two nearly identical vectors. One of the two vectors contains the complement to the normal base, and the other contains the complement to the mutated base. If the last base in the oligo is not complementary to the target, it will not hybridize, the padlock will not circularize, the polymerase will not progress continuously around the loop and no fluorescent signal will be detected. Consequently, the 'mutant' oligo will only be detected if it hybridizes completely with the mutant target, and the 'normal' oligo will only be seen if it hybridizes completely with a normal target.
- each RCA oligo In order to differentiate between the normal and mutant RCA vector products, each RCA oligo also has a short unique sequence incorporated in its non-binding region. Oligos with the same sequences as these sites are included in the reaction mixture; each sequence is labeled with a particular fluorophor (e.g., green for the normal and red for the mutant). As each loop is replicated, the sequence produced is complementary to the original loop, and therefore complementary to that of the labeled oligos. These labeled oligos hybridize to the RCA reaction products from their respective vector, and the 'normal' reaction products turn green and the 'mutant' products turn red.
- a particular fluorophor e.g., green for the normal and red for the mutant
- the number of targets examined simultaneously is limited only by the number of different fluorophors available.
- the technology for detecting products resulting from a PCR or other DNA amplification process allows differentiation among multiple targets.
- the polymerase is impeded by the interaction of the padlock probe with the target DNA as it replicates the loop. However, this severely limits the effectiveness of RCA. As the polymerase replicates the padlock circle, the target DNA becomes coiled within the ring and physically blocks polymerase activity. This blockage is far greater in fixed cells, in which the DNA is st conta ne n t e c romat n structure. e wor s we on DNA that has been extracted from cells and purified, it does not been work well in situ.
- RNA or DNA is extremely desirable, since it would allow specific mutations to be tracked throughout a diseased tissue, provide information on genetic changes and gene expression involved in initiation and progression of various conditions.
- DNA Detection As a first step in the process of performing DNA RCA in situ, cells and chromosomes are fixed to solid surfaces such as a microscope slide or cover slip by procedures well known in the art. Whole blood is cultured generally for 48 h after which Colcemid is added to arrest the cells in metaphase. Colcemid disrupts the mitotic spindle causing the cells to stop cycling in metaphase. The cultures are then harvested 4 h later by treatment with hypotonic solution such as 0.075 M KC1 followed by three fixations in methanol: acetic acid (3:1 v/v). The fixed cells are then dropped onto a solid surface such as 24 x 60 mm coverslips where they are air dried and stored at room temperature. The fixed cells are generally used within 24 hours of fixation in the procedures of this invention.
- the present invention is directed to a means to perform in situ
- RCA based on target preparation prior to hybridizing the oligo that allows commercially available DNA polymerases to be used for in situ RCA.
- the procedure of the present invention is based on avoiding the use of high processivity enzymes by preparing the target strand more effectively.
- the article by Nilsson, et al. (8) describes an RCA technique that requires a particular DNA polymerase, from the Bacillus subtilus phage ⁇ 29 to be successful. Any DNA polymerase with strand displacement ability will work with this invention.
- Endonuclease Digestion is an enzyme that makes a nick in both strands of double stranded DNA. The endonuclease digestion is carefully chosen to put a nick in the DNA strand to which the padlock probe will hybridize. Any restriction endonuclease will suffice so long as it is target specific. It is important to make the nick as close as possible to the binding site of the RCA vector without actually being within the vector's binding site. Also, the nick on the strand of DNA to which the vector binds must be 5' to the binding site. Each gene, mutation or region must be predigested by a particular endonuclease that is specifically chosen to make a nick in the appropriate place. Selection or an endonuclease is routine experimental work to one of ordinary skill in the art.
- exonuclease Digestion is an enzyme that digests double stranded
- Exonuclease HI will proceed from a nick in a DNA strand until it comes to a place where the DNA is already single stranded, and then it stops. Since the endonuclease digest makes nicks in both strands of the DNA, the exonuclease will digest both strands at the same time until a series of interlocking single stranded products remain. Careful choice of the restriction endonuclease cutting site as described above results in the target DNA sequence always remaining following exonuclease IH digestion.
- the RCA vector is then hybridized to the DNA template.
- This template can be, but is not limited to, eukaryotic, prokaryotic, viral, chromomosomal, mitochondrial or chloroplast DNA. Any type of nucleic acid can serve as a target. Hybridization and ligation occur simultaneously; those vectors that are not hybridized to their correct targets will not be ligated.
- the unbound/unligated target is washed off the slide on w ic e reac on s e ng per orme . e ro ng par o t e reac on s done as by procedures well known in the art.
- Heat stable polymerases find use in the methods of the invention as they allow the reactions to proceed at higher temperatures that are more conducive to target specificity.
- RNA may be detected by RCA in solution, on slides and in paraffin sections.
- the slides are generally prepared by centrifuging live cells suspended in phosphate-buffered saline onto glass slides followed by fixation in ethanol for time sufficient to fix the RNA to the slide, generally around 5 minutes.
- the choice of ethanol appears to be critical as the reaction does not work with methanol or acetic acid.
- the timing of the fixation step is not critical.
- the cells prepared for RNA detection by RCA are not treated with restriction enzymes or exonuclease IH, nor are they heat denatured.
- T4 RNA ligase (Epicenter) is used for the ligation of the probes to the RNA.
- the slides are stained with, e.g. Acridine Orange (AO) at room temperature, rinsed in buffer and sterile water at room temperature, then mounted in buffer and stained with DAPI.
- AO Acridine Orange
- Example 1 [0055] la. Target preparation Two cell lines were used in these experiments. One was a human lymphoblastoid (HLB) line (Coriell Cell Depository) putatively normal with regard to karyotype and gene expression. The other was a Molt-4 lymphoid cell line (available from the American Type Culture Collection) derived from a patient with acute lymphoblastoid leukemia. HLB cells were expected to have two normal copies of the Tp53 gene, and to be normal with regard to Tp53 expression. Molt-4 cells are reported by ATCC to express no normal Tp53, and to have one normal and one or more abnormal copies of the Tp53 gene, in which there is a G->A transition in codon 248 of exon 7.
- HLB human lymphoblastoid
- Molt-4 lymphoid cell line available from the American Type Culture Collection
- Cells were prepared for DNA detection by first incubating in a hypotonic solution (0.075 M KC1) for 30 min at 37°C followed by three fixations in methanol:acetic acid (3:1 v/v) and dropped on clean glass microscope slides. Fixed cells on slides were covered with 50 ⁇ l of onuc ease ⁇ g m , oc e un er a g ass covers p. es were incubated 1 hr at 37°C then rinsed with sterile water. Restriction enzymes were used to cut approximately 20 base pairs either 3 ' or 5' of the sequence of interest, Tp53 in this case.
- Either Afl HI or Bbs I were applied for 12 hours at 37°C.
- Cells were treated with Exonuclease HI (1.3U/ ⁇ l, Life Technologies) in IX exonuclease IH Buffer (50mM Tris, pH 8.0, 5mM MgCl 2 , lmM DTT), then incubated 1 hr at 37°C and rinsed with sterile water.
- the RCA reaction mixture consisted of 4 ⁇ M of T7 primer, 200 ⁇ M of each DNTP(Roche), either 63nM digoxigenin-11-dUTP (Roche) or 63nM biotin-dUTP (Roche), 2 Units ThermoSequenase DNA Polymerase with Pyrophosphatase (USB) and IX ThermoSequenase buffer which was added to the slide which was then covered with a coverslip, sealed with rubber cement and heated 12 hr at 54°C. Slides were washed in 2XSSC at 45°C for 5 minutes, 1XPBS at 45°C for 5 minutes and rinsed in sterile water at room temperature.
- Anti-digoxigenin-fluorescein antibody or Texas Red Avidin was incubated on the slide at 37°C for 10 minutes and washed 2x 5 min in 1XPBS at room temperature. Slides were mounted in 4',6'-diamido-2-phenylindole (DAPI) in anti-fade and viewed with an Axiophot Fluorescence Microscope (Zeiss).
- DAPI 4',6'-diamido-2-phenylindole
- TAATACGACTCACTATAGGG [SEQ ID NO:l] and also contained a separate promoter sequence used to hybridize a fluorochrome-tailed o gonuc eot e nc u e n t e react on to ts complementary amplified sequence.
- the SP6 promoter (ATTTAGGTGACACTATAG) [SEQ ID NO:2] was used to bind to the reaction products from the mutant probe, GTTCATGCCGCCCtttttttTATTTAGGTGACACTATAGtttttttCCCTATAG TGAGT CGTATTAtttttttttGGTGAGGATGGGCCTCT) [SEQ ID NO:3] and the T3 promoter (ATTAACCCTCACTAAAG) [SEQ ID NO:4]was used to bind to the reaction products of the normal probe
- Thymidines are used as spacers in the SP6 and T3 sequences and are indicated by lower-case letters.
- the procedure for ligation and rolling circle amplification are similar to the procedure for a single color reaction. Differences include ligating 0.4 ⁇ M of each probe (mutated and normal) as well as incorporating lO ⁇ M of fluorochrome tailed oligonocleotide (T3 and SP6) in place of digoxigenin-dUTP in the RCA reaction.
- the tailed oligonucleotides were used to obtain different colors for the mutant and normal Tp53 gene sequences. Fluorochrome tailing was achieved with a lO ⁇ M solution of the promoter oligonucleotide with digoxigenin-dUTP (T3 oligo) or biotin-dUTP (SP6 oligo) (lOOnM, Roche) using Terminal Deoxynucleotidyl Transferase (1.5U/ ⁇ l, Life Technologies), and IX Terminal Deoxynucleotidyl Transferase buffer. The reaction was incubated 1 hr at 37°C
- RNA Detection [0058] 2a. Qualitative detection: RCA using the human T ⁇ 53 mRNA complementary probe
- T4 RNA ligase (Epicenter, 20 Units) was used for the ligation under identical conditions as described above. The slides were stained for 3 minutes with 10 ⁇ g/ml Acridine Orange (AO), rinsed in buffer and sterile water at room temperature, then mounted in buffer and stained with DAPI.
- AO Acridine Orange
- Probe sequences for the RNA detection were: keratin 10,
- Figure 1 shows a flow diagram of DNA RCA in situ.
- Pretreatment prior to in situ RCA detection of the Tp53 gene is required. Restriction enzymes were used to cut approximately 20 base pairs either 3' or 5' of the probe binding site. Afl LU was used for digestion 5' of the binding site, and Bbs I was used for digestion 3' of the target. Cells were then treated with Exonuclease IH, which digests DNA 3' -5' starting with 3' hydroxyl left by the endonuclease, resulting in staggered single stranded DNA. The DNA strand remaining following Afl HI digestion in this case is the complement to the 'sense' probe sequence. DNA remaining following Bbsl digestion is the complement to the 'antisense' probe sequence.
- Bbs I digestion constitutes a negative control for the RCA process using the sense probe
- Afl IH constitutes a negative control for the antisense probe.
- the two ends of the sense probe create an incomplete circle as they anneal to the complementary site on the DNA digested with Bbs I.
- the DNA strand digested with Afl HI is complementary to the sense probe and allows it to anneal and ligate, completing the circle and locking the probe onto the target.
- Targets other than Tp53 may require different endonucleases.
- DNA was cut with a restriction endonuclease, then the double helix was rendered single-stranded by digestion with exonuclease IH. Padlock probe hybridization and RCA followed. Endonuclease digestion was carefully selected to place a cut within approximately 20 bases of the probe's binding site but without actually being within that site.
- the substrate for Exonuclease HI is double-stranded DNA, which it digests from a 3' end leaving a single strand of DNA in its wake, proceeding until it reaches a region where the DNA is already single stranded (9, 10). Thus, the entire genome will still be represented following exonuclease digestion, but for any given locus only one of the two strands will be present.
- Success of the RCA reaction depended on the probe's target sequence remaining intact following digestion. To ensure that the target strand was not removed by the exonuclease treatment, the endonuclease had to cut 5' of the site on the strand to which the probe bound, and a second cut must not occur too near the 3' end on the target strand.
- Identical RCA reactions were performed with two probes that were complementary to the Tp53 gene present in two copies in the target cells. One of the probes was complementary to the coding strand, the other to the non-coding strand. In one set of reactions the target strand was cut 3' to the binding site, and in the other set the cut was 5' to the site.
- reaction products were labeled by incorporating a hapten-conjugated nucleotide (either biotin or digoxigenin) in the reaction mixture.
- the results were detected by a subsequent treatment with a fluorochrome-conjugated (Texas Red or uorescem an o y o e ap en, pro uc ng fluorescent labeled signals at the site of the reaction.
- fluorochrome-conjugated Texas Red or uorescem an o y o e ap en, pro uc ng fluorescent labeled signals at the site of the reaction.
- reactions with 5 'nicks successfully produced labeled signals whereas reactions with nicks 3' to the target never produced signals.
- the sequences of two bacteriophage promoters were incorporated into the RCA probes. Because the T7 promoter primer is incorporated into the probes to initiate the RCA reaction, we incorporated either the T3 (for the normal sequence probe) or SP6 (for the mutant sequence probe) bacteriophage promoter sequences into the RCA probes. Oligomers corresponding to the two promoter sequences were then included in the RCA reaction and were differentially labeled, T3 with digoxigenin and SP6 with biotin. During the RCA reaction, each replication of the probe produces a single stranded sequence complementary to the promoter sequence contained within the probe.
- T3 or SP6 oligomers should hybridize to these sites as they are produced, labeling each product with either digoxigenin or biotin.
- Amplification products of Tp53 in Molt-4 cells were detected using a fluorescein-co ⁇ jugated anti-digoxigenin antibody and Texas Red-conjugated avidin to produce green and red signals at the sites of the normal and mutant alleles, respectively ( Figure 3).
- Figure 3 shows allele discrimination in a Molt-4 cell in which a single nucleotide (G to A) difference in two alleles of the Tp53 gene was detected by RCA in situ as discussed above. Two probes were used, which differed in that the 3' terminus of the normal and the mutant versions were complementary to the normal (green signal) and the mutant (red signal) sequences, respectively.
- the binding sequences of the mutant- complement and normal-complement probes differ by only a single base. Whereas this difference is placed so as to prohibit the two ends of the probe from being ligated and amplified should they anneal to the incorrect site mutant to norma s te or t e reverse , t s nsufficient to stop such mis- binding from occurring at all loci. Some fraction of the time, this mis- hybridization will occur, and while improperly-bound probe will be washed away prior to amplification, no signal will be produced at that site. Thus, for simultaneous 2-probe binding, to prevent such false negatives each probe should be hybridized, ligated and washed off sequentially.
- the wash step will not remove properly bound and ligated probe, but it will remove unligated material. This process will ensure the maximum possible efficiency of detection. Even with this precaution, efficiencies for two color detection were considerably lower than for single color RCA (approximately 30% as compared to greater than 90%). We attribute this to the method of fluorescence labeling, as it is similarly inefficient when used for only single color detection.
- RNA detection By designing an RCA probe's binding site to be complementary to a transcribed rnRNA sequence, gene expression could also be detected.
- the method of cell fixation for RNA detection was considerably more important than for DNA detection.
- various methods of cell preparation, including conventional acid: alcohol fixation and alcohol fixation yielded similar results for DNA-based RCA in situ, routine detection of RNA was made possible by centrifuging the cells onto slides in culture media followed by an alcohol wash.
- a probe with a 30 base binding site complementary to a transcribed region of the Tp53 gene was used to determine the presence of Tp53 mRNA in Molt-4 and HLB cell lines. Unlike HLB cells, Molt-4 cells produce no normal Tp53 transcripts (11).
- Single-color RCA was performed both before and after treatment of fixed cells with RNase.
- HLB and Molt-4 cells were also stained with Acridine Orange (AO) following all treatments prior to the RCA step to ensure that the results of the RCA reaction corresponded with the actual status of RNA in the cells.
- AO staining detected RNA prior to, but never following, RNase treatment.
- RCA performed on HLB cells that had not been treated with RNase showed considerable labeling in the cytoplasmic region surrounding the nucleus ( Figure 4).
- Figure 4 shows mRNA detection in normal HLB cells (A)
- Molt-4 cells (B) stained with Acridine Orange (AO), which labels single stranded nucleic acid (RNA) red and double strari ⁇ e ⁇ " nucleic 'ac d" (nuclear DNA) yellow. Panels A and B demonstrate the presence of RNA in each of the cell types. RCA was performed on replicate cell preparations using a probe with 3' and 5' DNA binding site complementary to the probe described in Figure 3. (C) The green fluorescence signal surrounding the nuclei of the HLB cells demonstrates the presence of Tp53 transcript detected by RCA. (D) No such signal is seen in the Molt-4 cells, demonstrating the lack of normal Tp53 transcript.
- AO Acridine Orange
- RCA probes were constructed to bind to the mRNA of several genes known to be radiation dose responsiveas shown at the web site rex.nci.nih.gov/RESEARCH basic/lbc/patent/web6kinduced including Tp53, human DEAD-box protein p72, vimentin, keratin 10 and glutathione S- transferase theta 2 (GSTT2). HLB cells were exposed to 137 Cs gamma rays at doses up to 2 Gy, then fixed and evaluated by RCA.
- Use of DNA ligases to join single stranded fragments of DNA hybridized to an RNA target has been described in the literature (13).
- As a negative control each RCA reaction was also run without ligase, the results of which were used to normalize the results of the experiments.
- a probe complementary to untranscribed alpha satellite DNA was used as an additional negative control. Neither negative control produced a response signal; results are shown in Figure 5.
- Figure 5 shows radiation dose response curves for normal HLB cells that were irradiated, left in culture medium for 2 hours, and then fixed and analyzed.
- T4 RNA ligase 20 U in 4 ⁇ l lOx buffer, Epicentre Technologies
- Each experiment was replicated n ⁇ ni a u , .
- Fifty to 150 cells were analyzed per data point; error bars represent +/- SEM.
- Cells were analyzed by measuring mean pixel intensity of green fluorescence using IPLabs image analysis software (Scanalytics Inc.).
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002239679A AU2002239679A1 (en) | 2000-12-20 | 2001-12-18 | Rolling circle amplification detection of rna and dna |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US25721600P | 2000-12-20 | 2000-12-20 | |
US60/257,216 | 2000-12-20 |
Publications (3)
Publication Number | Publication Date |
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WO2002050310A2 WO2002050310A2 (en) | 2002-06-27 |
WO2002050310A9 true WO2002050310A9 (en) | 2002-11-28 |
WO2002050310A3 WO2002050310A3 (en) | 2004-03-04 |
Family
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Application Number | Title | Priority Date | Filing Date |
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PCT/US2001/049780 WO2002050310A2 (en) | 2000-12-20 | 2001-12-18 | Rolling circle amplification detection of rna and dna |
Country Status (3)
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US (1) | US6783943B2 (en) |
AU (1) | AU2002239679A1 (en) |
WO (1) | WO2002050310A2 (en) |
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US7141371B2 (en) * | 2002-09-06 | 2006-11-28 | State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Methods for detecting and localizing DNA mutations by microarray |
ATE483032T1 (en) | 2005-04-12 | 2010-10-15 | Olink Ab | METHOD FOR PRODUCING OLIGONUCLEOTIDES |
CN101238221B (en) * | 2005-04-12 | 2011-11-16 | Novia公司名下的现场Rcp公司 | Ring probe and its uses in identifying biomolecule |
US7449297B2 (en) | 2005-04-14 | 2008-11-11 | Euclid Diagnostics Llc | Methods of copying the methylation pattern of DNA during isothermal amplification and microarrays |
WO2006122215A2 (en) * | 2005-05-10 | 2006-11-16 | State Of Oregon Acting By & Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Methods of mapping polymorphisms and polymorphism microarrays |
JP5331476B2 (en) * | 2005-06-15 | 2013-10-30 | カリダ・ジェノミックス・インコーポレイテッド | Single molecule array for genetic and chemical analysis |
US7960104B2 (en) | 2005-10-07 | 2011-06-14 | Callida Genomics, Inc. | Self-assembled single molecule arrays and uses thereof |
US20070168197A1 (en) * | 2006-01-18 | 2007-07-19 | Nokia Corporation | Audio coding |
SG10201405158QA (en) * | 2006-02-24 | 2014-10-30 | Callida Genomics Inc | High throughput genome sequencing on dna arrays |
EP1994180A4 (en) | 2006-02-24 | 2009-11-25 | Callida Genomics Inc | High throughput genome sequencing on dna arrays |
GB0621361D0 (en) * | 2006-10-26 | 2006-12-06 | Fermentas Uab | Use of DNA polymerases |
US7910302B2 (en) | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
US20080221832A1 (en) * | 2006-11-09 | 2008-09-11 | Complete Genomics, Inc. | Methods for computing positional base probabilities using experminentals base value distributions |
US20090111705A1 (en) | 2006-11-09 | 2009-04-30 | Complete Genomics, Inc. | Selection of dna adaptor orientation by hybrid capture |
US7897344B2 (en) * | 2007-11-06 | 2011-03-01 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs |
US20090263872A1 (en) * | 2008-01-23 | 2009-10-22 | Complete Genomics Inc. | Methods and compositions for preventing bias in amplification and sequencing reactions |
US8518640B2 (en) * | 2007-10-29 | 2013-08-27 | Complete Genomics, Inc. | Nucleic acid sequencing and process |
US8298768B2 (en) * | 2007-11-29 | 2012-10-30 | Complete Genomics, Inc. | Efficient shotgun sequencing methods |
US8415099B2 (en) | 2007-11-05 | 2013-04-09 | Complete Genomics, Inc. | Efficient base determination in sequencing reactions |
WO2009061840A1 (en) * | 2007-11-05 | 2009-05-14 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors employing selective methylation |
US8592150B2 (en) | 2007-12-05 | 2013-11-26 | Complete Genomics, Inc. | Methods and compositions for long fragment read sequencing |
WO2009097368A2 (en) | 2008-01-28 | 2009-08-06 | Complete Genomics, Inc. | Methods and compositions for efficient base calling in sequencing reactions |
WO2009132028A1 (en) * | 2008-04-21 | 2009-10-29 | Complete Genomics, Inc. | Array structures for nucleic acid detection |
US9524369B2 (en) | 2009-06-15 | 2016-12-20 | Complete Genomics, Inc. | Processing and analysis of complex nucleic acid sequence data |
BR112013020773B1 (en) * | 2011-02-15 | 2023-01-24 | Leica Biosystems Newcastle Limited | METHOD FOR LOCALIZED IN SITU DETECTION OF RNA, METHOD FOR DETERMINING THE PRESENCE AND LOCATION OF A GENETIC SEQUENCE AND METHOD FOR IN SITU LOCATION AND/OR DETECTION OF A SPECIFIC RNA |
EP2744916A4 (en) | 2011-07-13 | 2015-06-17 | Primeradx Inc | Multimodal methods for simultaneous detection and quantification of multiple nucleic acids in a sample |
US20130157259A1 (en) * | 2011-12-15 | 2013-06-20 | Samsung Electronics Co., Ltd. | Method of amplifying dna from rna in sample and use thereof |
US10208339B2 (en) * | 2015-02-19 | 2019-02-19 | Takara Bio Usa, Inc. | Systems and methods for whole genome amplification |
WO2018107054A1 (en) | 2016-12-09 | 2018-06-14 | Ultivue, Inc. | Improved methods for multiplex imaging using labeled nucleic acid imaging agents |
US20190382838A1 (en) * | 2018-06-01 | 2019-12-19 | The Trustees Of The University Of Pennsylvania | Methods For Single-Molecule Fluorescence Amplification Of RNA |
AU2019371382A1 (en) * | 2018-10-31 | 2021-05-20 | The Board Of Trustees Of The Leland Standford Junior University | Methods and kits for detecting cells using oligonucleotide conjugated antibodies |
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US4889799A (en) * | 1984-02-17 | 1989-12-26 | Fred Hutchinson Cancer Research Center | Reagent kit producing shortened target DNA segments usable in sequencing large DNA segments |
CA2218875C (en) * | 1991-07-23 | 2000-11-07 | The Research Foundation Of State University Of New York | Improvements in the in situ pcr |
US5543297A (en) * | 1992-12-22 | 1996-08-06 | Merck Frosst Canada, Inc. | Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity |
US5854033A (en) * | 1995-11-21 | 1998-12-29 | Yale University | Rolling circle replication reporter systems |
WO1997019193A2 (en) * | 1995-11-21 | 1997-05-29 | Yale University | Unimolecular segment amplification and detection |
AU3567099A (en) * | 1998-04-16 | 1999-11-01 | Packard Bioscience Company | Analysis of polynucleotide sequence |
CA2342837A1 (en) * | 1998-09-15 | 2000-03-23 | Yale University | Artificial long terminal repeat vectors |
-
2001
- 2001-12-18 US US10/032,017 patent/US6783943B2/en not_active Expired - Fee Related
- 2001-12-18 AU AU2002239679A patent/AU2002239679A1/en not_active Abandoned
- 2001-12-18 WO PCT/US2001/049780 patent/WO2002050310A2/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
AU2002239679A1 (en) | 2002-07-01 |
WO2002050310A3 (en) | 2004-03-04 |
WO2002050310A2 (en) | 2002-06-27 |
US6783943B2 (en) | 2004-08-31 |
US20020177142A1 (en) | 2002-11-28 |
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