WO1991015601A1 - Reaction en chaine de polymerase specifique de reproduction d'a.r.n. modifiee - Google Patents

Reaction en chaine de polymerase specifique de reproduction d'a.r.n. modifiee Download PDF

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WO1991015601A1
WO1991015601A1 PCT/US1991/002211 US9102211W WO9115601A1 WO 1991015601 A1 WO1991015601 A1 WO 1991015601A1 US 9102211 W US9102211 W US 9102211W WO 9115601 A1 WO9115601 A1 WO 9115601A1
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sequence
dna
pcr
segment
primer
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PCT/US1991/002211
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Alan R. Shuldiner
Jesse Roth
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The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
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Priority to AU76536/91A priority Critical patent/AU7653691A/en
Publication of WO1991015601A1 publication Critical patent/WO1991015601A1/fr

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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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the present invention relates to a method for detecting an RNA sequence. More specifically, the present invention relates to a method of amplifying an RNA sequence using a modification of the polymerase chain reaction.
  • PCR polymerase chain reaction
  • this technique When coupled with reverse transcription (RT-PCR) , this technique can detect as few as 1 to 100 copies of a specific RNA from single cells or small numbers of cells. [Kawasaki et al., Proc. Natl. Acad. Sci. U.S.A. 85, 5698 (1988); Rappolee et al., Science 241, 708 (1988); Rappolee et al., Science 241, 1823 (1988); Sarkar et al., Science 244, 331 (1988); and Froh an et al., Proc. Natl. Acad. Sci. U.S.A. 85, 8998 (1988)]. Unfortunately, the extraordinar sensitivity of this technique presents one of its severe shortcomings, false positives resulting from contamination with minute quantities of DNA [Kwok et al.. Nature 339, 237 (1989); Sakar et al.. Nature 343, 27 (1989)].
  • Potential sources of contaminating DNA may include:
  • exogenous sources such as cDNA, plasmid DNA, or DNA fragments amplified in previous PCRs (i.e. carryover) .
  • RT-PCR was recently used to detect small quantities of Xenopus insulin mRNAs in unfertilized eggs and early embryos.
  • numerous precautions were taken to exclude contamination of Xenopus insulin cDNAs which had been previously cloned in our laboratory [Shuldiner et al., J. Biol. Chem. 264,9428 (1989)], frequent false positives precluded meaningful ⁇ interpretation of the experiments.
  • RNA template-specific PCR and modified RS-PCR, provides methods of detecting minute quantities of RNA without the problems of false positives associated with RT-PCR.
  • RS-PCR RNA template-specific PCR
  • modified RS-PCR provides methods of detecting minute quantities of RNA without the problems of false positives associated with RT-PCR.
  • the reduction in the frequency of false positives is achieved without sacrificing sensitivity obtained with conventional RT-PCR.
  • RNA sequence which reduces the number of false positives resulting from DNA contamination in the sample (i.e., previously cloned cDNAs, genomic DNA or carryover of DNA amplified in previous PCRs) .
  • the present method increases the accuracy of the procedure without sacrificing sensitivity.
  • the present invention relates to a method of detecting an RNA sequence.
  • the method comprises: i) reverse transcribing the RNA sequence from an oligonucleotide primer hybridized thereto which oligonucleotide primer (d 17 -t 30 ) comprises: a) on the 3 1 end thereof (segment d 17 ) , a nucleotide sequence complementary to a region of the RNA sequence to be detected; and b) on the 5* end thereof (segment t 30 ) , a unique random nucleotide sequence or tag whereby a single stranded DNA sequence is produced which has at its 5' end the unique sequence; ii) hybridizing, at a temperature high enough to preclude annealing of the d 17 segment of the d ⁇ 7 -t 30 primer to possible contaminating DNA, but low enough to allow annealing, an upstream oligonucleotide primer (U 30 ) , to a region of said DNA sequence
  • FIGURE 1 shows diagrammatically the RNA template-specific PCR method (RS-PCR) .
  • FIGURE 2 compares the sensitivity of conventional reverse transcriptase - PCR (RT-PCR) and novel RS-PCR when beginning with an RNA template.
  • FIGURE 3 compares conventional RT-PCR and novel RS-PCR when DNA rather than RNA is used as a starting template to mimic DNA contamination.
  • FIGURE 4 shows the effect of changing the nucleotide sequence of the unique segment of oligonucleotide primer d 2 o _ 2 i•
  • FIGURE 5 shows schematically the modified RNA template-specific PCR.
  • FIGURE 6 compares conventional RT-PCR and modified RS-PCR.
  • RT primer d 7t 30 PCR primers t 30 and u 30 (lanes 1-4)
  • RT primer d 30 t PCR primers d 30 and u 30 (lanes 6-9) .
  • Lane 0 is a Haelll digest of PhiX174 DNA, while lane 5 is RS-PCR in the absence of any template.
  • FIGURE 7 shows PCR carryover contamination is ignored with modified RS-PCR. Lanes 1 and 3; RT primer d 17 -t 30 , PCR primers u 30 and t 30 . Lanes 2 and 4; RT primer d 16 t' 30 , PCR primers u 30 and t' 30 .
  • FIGURE 8 shows the region of Xenopus insulin RNA that was used as the target RNA to test the modified RS-PCR procedure.
  • Reverse transcription primer d 17 -t 3 o contained a 17 base sequence at its 3' end (segment d 17 ) that was complementary (antisense) to a region of Xenopus insulin RNA in the 3' untranslated region (nucleotides 404-420), and 30 bases at its 5 1 end (segment t 30 ) that were unique in sequence.
  • Upstream primer u 30 is identical (sense) to Xenopus insulin RNA in the coding region (nucleotides 59-88) .
  • RNA template-specific PCR and modified RS-PCR, relates to a targeted amplification method which distinguishes RNA in the sample from contaminating DNA and amplifies only sequences derived from RNA. Minute quantities of cDNA, plasmid DNA or carryover DNA amplified in previous PCRs can be important sources of contamination when using conventional RT-PCR.
  • the present invention reduces the number of false positives obtained as a result of contaminating DNA.
  • the present invention obviates the necessity of choosing a target RNA sequence which spans an intron in order to distinguish the reverse transcribed DNA from contaminating genomic DNA.
  • the modified RS-PCR eliminates the need for removal of the primer after reverse transcription, such as by ultrafiltration.
  • a first oligonucleotide primer designated d 20 -t 21 in Figure 1 (advantageously, of about 41 nucleotides) is hybridized to the RNA sequence to be detected.
  • Primer d 2 o-t 2 ⁇ comprises on the 3• end, a nucleotide sequence (advantageously, about 20 nucleotides) complementary to the 3 1 end of the RNA sequence whose presence is to be detected (segment d 2 o) and on the 5' end, a unique random nucleotide sequence or tag (advantageously, about 21 nucleotides) (segment t 2 ) . While the 3 1 end of the primer hybridizes to the RNA sequence, the 5' end of the primer remains unhybridized as no complementary sequence exists within the sample.
  • primer d 20 -t 2 has been hybridized to the 3 1 end of the RNA sequence, reverse transcriptase is used to extend the primer.
  • the resulting single -(-) stranded DNA segment is thus tagged at its 5' end with the unique sequence t 21 of original primer d 20 -t 21 .
  • This unique 5' sequence (t 21 ) distinguishes between DNA generated from the RNA-template and possible contaminating DNA. It is preferable for the unique sequence to be composed of approximately equal amounts of each nucleotide (i.e. about 25% of each nucleotide) .
  • sequence which is unlikely to have significant secondary structure, and does not contain significant complementarity at its 3• end with the 3' end of the upstream primer (for example, primer u 2 ⁇ ) .
  • the sequence can also, be selected so as to contain a convenient restriction enzyme recognition site if desired.
  • One skilled in the art can easily generate by computer appropriate sequences, 5•-GACAAGCTTCAGGTAATCGAT-3' and 5*-CCGAATTCTGTAGTCCGTCA-3* being two examples.
  • excess primer d 20 -t 21 is removed by ultrafiltration through a Centricon 100 device (Amicon, Danvers, MA) or similar device.
  • the DNA segment resulting from the previous step is amplified using the PCR technique (see U.S. Patents 4,683,202 and 4,683,195).
  • Two oligonucleotide primers designated u 21 and t 21 in Figure 1 are utilized to amplify the DNA.
  • Upstream oligonucleotide primer u 21 (advantageously, about 21 nucleotides) comprises a nucleotide sequence complementary the single stranded DNA segment produced in Step 1, a predetermined distance upstream from primer d 20 t 21 .
  • Oligonucleotide primer t 2 ⁇ (advantageously, about 21 nucleotides) comprises the unique nucleotide sequence with which the segment of DNA was tagged during reverse transcription. The two primers are added to the sample and the PCR is carried out.
  • primer u 21 which is complementary to a region of the single stranded DNA segment produced in Step 1, a predetermined distance upstream from primer d 20 t 21 , hybridizes thereto and is extended therefrom creating a complementary strand of DNA which includes at its 3• end a sequence complementary to the unique sequence.
  • Primer t 21 is not utilized in the first PCR cycle since no complementary sequence is present in the sample.
  • primer t 2 ⁇ is used in the second PCR cycle and all cycles thereafter.
  • the double stranded DNA segment resulting from the first PCR cycle is denatured prior to the second PCR cycle.
  • primer u 2 ⁇ which is complementary to the 3 1 end of the single - (-) stranded DNA hybridizes thereto and is extended therefrom.
  • primer t 21 hybridizes to its complementary sequence at the 3 ' end of the single - (+) strand of DNA and is extended therefrom. All DNA synthesis occurs in the 5 1 to 3' direction.
  • the modified RS-PCR method of the present invention is shown schematically in Figure 5.
  • the modified RS-PCR method eliminates the need to remove the first oligonucleotide primer, designated d 7 -t 30 in Figure 5, by selecting oligonucleotide primers d 17 -t 30 , t 30 and d 30 , so that differential hydridization occurs under the PCR conditions.
  • the primers are selected so that the d 7 -t 30 primer and the d 30 and the t 30 primers anneal under different temperatures.
  • a first oligonucleotide primer designated d 17 -t 30 in Figure 5 (advantageously, of about 47 nucleotides) is hybridized to the RNA sequence to be detected.
  • Primer d 17 -t 30 comprises on the 3' end, a nucleotide sequence (advantageously, about 17 nucleotides) complementary to the 3' end of the RNA sequence whose presence is to be detected (segment d ⁇ 7 ) , and on the 5* end, a unique random nucleotide sequence or tag (advantageously, about 30 nucleotides) (segment t 30 ) .
  • the primer should be selected so that the length of the d segment is such that it will not anneal efficiently to any DNA contaminants at the elevated annealing temperatures used in Steps 2 and 3.
  • One skilled in the art can easily generate by computer suitable d 17 -t 3 o primers including, for example, 5 1 - gaacatcgatgacaagcttaggtatcgatatgatggaattgccttga-3' and 5*-cttatacggatatcctggcaattcggacttgcatgatggaattgcc-3' .
  • reverse transcriptase is used to extend the primer thereby creating a single -(-) stranded DNA segment which is tagged at its 5* end with the unique sequence, the t 30 segment, of original primer.
  • This unique 5 1 sequence as with the RS-PCR method, distinguishes between DNA generated from the RNA- template and possible contaminating DNA.
  • oligonucleotide primer designated u 30 in Figure 5 hybridizes to the single stranded DNA generated in Step 1 a predetermined distance upstream from primer d 17 -t 30 , and is extended therefrom creating a complementary strand of DNA which includes at its 3* end a sequence complementary to the unique sequence.
  • the primer u 30 comprises a nucleotide sequence complementary to the single stranded DNA segment produced in Step 1, a predetermined distance upstream from primer d 17 t 30 .
  • the annealing stage of the PCR cycle is carried out at a temperature high enough to preclude annealing of the d 17 segment of the reverse transcription primer d 7 -t 30 a to contaminating DNA, but low enough to allow annealing of PCR primer u 30 , for example temperatures of 42" C or greater.
  • the double stranded DNA segment resulting from the first PCR cycle is denatured prior to the second PCR cycle.
  • primer u 3 o which is complementary to the single - (-) stranded DNA hybridizes thereto and is extended therefrom.
  • a second oligonucleotide primer designated t 30 in Figure 5 (advantageously, about 30 nucleotides) is added to the sample.
  • the primer t 30 comprises the unique nucleotide sequence with which the segment of DNA was tagged during reverse transcription. When the primer is added to the sample it hybridizes to its complementary sequence at the 3 1 end of the single - (+) strand of DNA and is extended therefrom.
  • the annealing stage of all PCR cycles is conducted at a temperature high enough to preclude annealing of the d 17 segment of the reverse transcription primer d 17 -t 30 a to contaminating DNA, but low enough to allow annealing of PCR primers u 3 o and t 30 .
  • RS-PCR sequences derived from RNA that are tagged with the unique sequence (t 30 ) during reverse transcription (step 1) are amplified preferentially during PCR (steps 2 and 3) .
  • the original RS-PCR method requires ultrafiltration after reverse transcription to remove excess RT primer [Shuldiner et al.. Gene 91, 139 (1990) ] .
  • the modified RS-PCR cirumvents this step by increasing the length of the RT and PCR primers, and increasing the PCR annealing temperature.
  • the primers are selected so that the RT primer, d 17 -t 3 o, hybridizes to the RNA template under the reverse transcription conditions but does not hybridize to possible DNA contaminants under the PCR conditions.
  • the longer length of the u 30 and t 30 primers allows annealing to occur at an increased temperature, that is temperatures up to about 72" C. Annealing of the 17 base d 7 segment of the RT primer d 17 -t 30 occurs efficiently during reverse transcription at 37°C, but not at the higher PCR annealing temperature. Thus, when Steps 2 and 3 are carried out at a temperature of 42" C or greater (preferably 65° or greater), remaining d 17 -t 30 primer does not anneal to possible DNA contaminants while the u 30 and t 30 primers will anneal and be extended.
  • each cycle of PCR involves primer hybridization, extension to yield double stranded DNA and denaturation.
  • both the (+) and (-) strands of the DNA serve as templates from which a new strand of DNA is created. This leads to logarithmic expansion of the tagged segment of DNA.
  • Contaminating DNA lacks the unique nucleotide sequence.
  • the 3* end of the single - (-) strand of DNA serves as a template for primer u 21 or u 30 (but the 3' end of the -(+) strand can not act as template for unique primer t 2 ⁇ or t 3 o since there is no complementary sequence) .
  • This allows only linear amplification which, as one skilled in the art knows, does not produce enough DNA to result in a false positive when detecting the presence of the logarithmically amplified PCR product.
  • the criteria for selecting the unique sequence of the primer used for reverse transcription and subsequent PCR is that the sequence selected is not present in the sample i.e. is unique. Therefore, the sequence used can be changed periodically. Changing the unique sequence prevents amplification of carryover PCR products.
  • the methods of the present invention are particularly useful in a clinical laboratory setting where many samples and automation make careful laboratory hygiene more difficult.
  • the present invention is as sensitive as the well known PCR and RT-PCR procedures. Therefore, the small quantity of RNA needed is not affected. However, the present invention has the advantage of being more accurate.
  • a segment of Xenopus insulin RNA is amplified by the present methods. The methods are applicable to the amplification of other RNAs.
  • RNA Template-Specific Polymerase Chain Reaction Xenopus insulin mRNA was amplified using novel RS-PCR, which involves first reverse transcribing Xenopus pancreatic RNA using an oligonucleotide 41-mer as a primer (oligonucleotide d 2 o-t 2 ⁇ ) whose nucleotide sequence contained 20-bases at the 3*-end which were complementary to a region of Xenopus insulin mRNA (segment d 20 ) , and 21-bases at the 5*-end which consisted of a unique random sequence selected by computer or similar method (segment t 21 ) (FIGURE 1) followed by PCR amplification of the DNA segment.
  • oligonucleotide 41-mer oligonucleotide d 2 o-t 2 ⁇
  • RNA from Xenopus pancreatic tissue was prepared by the guanidinium isothiocyanate method [Chirgwin et al.. Biochemistry 18, 5294 (1979)].
  • RNA was reverse transcribed at 42"C for one hour in a 25 ⁇ l reaction mixture containing Tris-HCl (50 mM, pH adjusted to 8.7 at room temperature) , NaCl (100 mM) , MgCl 2 (6 mM) , dithiothreitol (10 mM) , dNTP's (1 mM each), RNasin (1 ⁇ l; Promega Biotec; Madison, WI) , oligonucleotide d 20 - t 21 (5•-GACAAGCTTCAGGTATCGATTTGCATGATGGAATTGCCTTG-3 » ; 0.5 ⁇ M) , and AMV-reverse transcriptase (10 units; Promega Biotec) .
  • oligonucleotide primer d 20 -t 21 was efficiently removed (>99.9%) using a Centricon 100 ultrafiltration device (Amicon; Danvers, MA) according to manufacturer's recommendations. Then PCR was performed using as primers oligonucleotide t 21 , a 21-mer containing the same unique nucleotide sequence as in segment t 21 of oligonucleotide d 20 -t 2 ⁇ and oligonucleotide u 21 , a 21-mer complementary to the first strand, 244 bp upstream from oligonucleotide 2 ⁇ « PCR amplification was performed in a 50 ⁇ l reaction volume containing Tris-HCl (10 mM, pH adjusted to 8.3 at room temperature), KC1 (50 mM) , MgCl 2 (1.5 mM), gelatin (0.01%), dNTP's (200 ⁇ M each), oligonucleotide t 21
  • the reaction mixture was covered with paraffin oil (approximately 50 ⁇ l) , heated to 94"C for 5 minutes, followed by PCR (45-60 cycles). Each cycle consisted of annealing (55'C, 1.5 min) , extension (72"C, 1.5 min) and denaturation (94"C, 1 min) except for the last cycle, in which the extension time was increased to 15 minutes to insure completeness of extension. Twenty microliters of the reaction mixtures were loaded onto a composite gel consisting of 1% agarose and 2% Nusieve GTG (FMC Bioproducts; Rockland, ME) in Tris-borate-EDTA buffer, electrophoresed, stained with ethidium bromide, and visualized by UV transillumination.
  • paraffin oil approximately 50 ⁇ l
  • PCR 45-60 cycles. Each cycle consisted of annealing (55'C, 1.5 min) , extension (72"C, 1.5 min) and denaturation (94"C, 1 min) except for the last cycle, in which the extension
  • RNA which had been reverse transcribed with oligonucleotide d 20 - t 21 and ultrafiltered, was subjected to either conventional RT-PCR (oligonucleotides d 20 and u 21 ) , or novel RS-PCR (oligonucleotides t 21 and u 21 ) .
  • PCR with either of these two oligonucleotide pairs resulted in similar sensitivity (FIGURE 2) .
  • Xenopus pancreatic RNA (1 ng) was reverse transcribed and ultrafiltered according to the methods of Example 1.
  • Example 3 Comparison of novel RS-PCR and conventional RT-PCR using a DNA template
  • novel RS-PCR was approximately 10 to 1000-fold less affected by the presence of DNA contaminants (i.e., Xenopus insulin cDNA) than conventional RT-PCR even after 60 cycles (FIGURE 3) .
  • Full-length Xenopus insulin cDNA (300 pg) was "reverse transcribed" with oligonucleotide d 20 -t 21 , excess oligonucleotide d 20 -t 2 ⁇ removed by ultrafiltration, and PCR (60 cycles) was accomplished as described in the above Examples.
  • Results of the conventional RT-PCR performed on serial ten-fold dilutions of the "reverse transcribed" and ultrafiltered Xenopus insulin cDNA using oligonucleotides d 20 and u 21 is shown in FIGURE 3, lanes 1-5.
  • Novel RS-PCR of identical serial ten-fold dilutions of "reverse transcribed" and ultrafiltered Xenopus insulin cDNA using oligonucleotides t 21 and u 21 is shown in lanes 6-10 of the same figure.
  • RNA that had been primed with oligonucleotide d 2 o-t 21 during RT should have been amplified during PCR.
  • detectable amplification was observed (lane 6 in Fig. 3) .
  • Example 4 Effect of changing the sequence of the unique segment t 2. of oligonucleotide d 20 - t 21 on conventional RT-PCR and novel RS- PCR.
  • oligonucleotide d 20 - t 21 on conventional RT-PCR and novel RS- PCR.
  • Xenopus pancreatic RNA (1 ng) was reversed transcribed with either oligonucleotide 41-mer d 20 -t 21 (FIGURE 4, lanes 1, 2, 3 and 7), or oligonucleotide 41- mer d 20 -t' 21 (5 » - CCGAATTCTGTAGTCCGTCATTGCAGATGGAATTGCCTTG-3•) (FIGURE 4, lanes 4-6) .
  • PCR 45 cycles was accomplished as described in the previous Examples using oligonucleotide pairs t 21 and u 21 (FIGURE 4, lanes 1 and 4), t' 21 (5'-CCGAATTCTGTAGTCCGTCA-3') and u 21 (FIGURE 4, lanes 2 and 5), d 20 and u 21 (FIGURE 4, lanes 3 and 6), or t 21 and u' 21 (5'-TGACCTTTCCAGCACTTATC-3') (FIGURE 4, lane 7) .
  • RNA that had been reversed transcribed with oligonucleotide d 20 -t 21 was amplified only when oligonucleotide t 21 was used during PCR, but not when an unrelated unique 21-mer (oligonucleotide t' 21 ) was used.
  • reverse transcription of Xenopus pancreatic RNA with oligonucleotide d 20 -t' 21 could only be amplified by the corresponding unique 21- mer, oligonucleotide t' 21 , and not by the unrelated random 21-mer, oligonucleotide t 21 .
  • oligonucleotide d 20 reverse transcription of Xenopus pancreatic RNA with oligonucleotide d 20 -t' 21
  • Oligonucleotides were synthesized on a Coder 300 automated DNA synthesizer (E.I. Du Pont Company;
  • Xenopus insulin (sense) RNA was prepared by ligating an 890 bp Xenopus insulin cDNA [Shuldiner et al., J. Biol. Chem. 264, 9428 (1989)] into pSP71 (Promega Biotec; Madison, WI) .
  • RNA polymerase Promega Biotec
  • T7 RNA polymerase was used for in vitro transcription to generate Xenopus insulin (sense) RNA.
  • the RNA was purified by oligo-dT cellulose chromatography (Bethesda Research Laboratories) . Only full-length RNA was retained by the column since the 3' end contained a long poly-A tail. The RNA was quantitated by UV absorbance at 260 nm. RNA was diluted to the appropriate concentration in water containing yeast tRNA (100 ⁇ g/ml) (Bethesda Research Laboratories) .
  • DNA templates used to demonstrate RNA specificity were either a double-stranded 890 bp Xenopus insulin cDNA insert [Shuldiner et al., J. Biol. Chem. 264, 9428 (1989)], or a 377 bp Xenopus insulin RS-PCR product that had been subjected to ultrafiltration with a Millipore-MC-100 device (Millipore; Bedford, MA) to remove excess primers. DNA templates were quantitated by comparison to a known quantity of a Haelll digest of PhiX174 (Bethesda Research Laboratories) after agarose gel electrophoresis.
  • Reverse transcription of serial ten-fold dilutions of Xenopus insulin RNA (10 7 to 10 4 copies) was accomplished at 37° C in a final volume of 20 ⁇ l containing KC1 (50 mM) , Tris-HCl (10 mM; pH 8.3 at 25" C) , MgCl 2 (1.5 mM) , gelatin (0.01 mg/ml), dNTPs (200 ⁇ M each) , RNasin (40 U; Promega Biotec) , AMV- reverse transcriptase (7 U; Promega Biotec) , and primer.d 17 -t 30 (0.5 ⁇ M) .
  • Primer d 17 -t 30 (Table I) was a 47-mer whose sequence contained 17 bases at its 3•-end that were complementary to a region of Xenopus insulin mRNA, designated segment d 17 , and 30 bases at its 5'-end that were unique in sequence, designated segment t 30 .
  • reverse transcription yields single-stranded DNA that contains a unique 30 base "tag" (segment t 30 ) at its 5 « end (FIGURE 5).
  • the second strand was synthesized during the first cycle of PCR in which 5 ⁇ l of the RT reaction mixture from step 1 was used directly in a final volume of 50 ⁇ l containing KC1 (50 mM) , Tris-HCl (10 mM; pH 8.3 at 25° C) , MgCl 2 (1.5 mM) , gelatin (0.01 mg/ml), dNTPs (200 ⁇ M each), upstream primer u 30 (0.5 ⁇ M) , downstream primer t 30 (0.5 ⁇ M) and Taq polymerase (1.5 U; Perkin Elmer-Cetus; Emeryville, CA) .
  • KC1 50 mM
  • Tris-HCl 10 mM; pH 8.3 at 25° C
  • MgCl 2 1.5 mM
  • gelatin 0.01 mg/ml
  • dNTPs 200 ⁇ M each
  • upstream primer u 30 0.5 ⁇ M
  • downstream primer t 30 0.5 ⁇ M
  • Upstream (sense) primer u 30 was a 30-mer corresponding to Xenopus insulin cDNA that was 347 bp upstream from the sequence corresponding to segment d ⁇ 7 , while downstream primer t 30 was a 30-mer whose sequence was identical to segment t 30 of RT primer d 17 -t 30 (see Table I) .
  • sequences derived from RNA that had been tagged with unique sequence (t 30 ) during reverse transcription were amplified logarithmically preferentially, while contaminating DNAs, lacking the unique tag, were not amplified logarithmically (FIGURE 5) [Shuldiner et al.. Gene 91, 139 (1990)].
  • PCR reaction mixture After covering the PCR reaction mixture with parafin oil (approximately 50 ⁇ l) , 35 cycles of PCR were performed, each cycle consisting of denaturation (94° C, 1 mih) and annealing/extension (70° C, 2 min) . In the first cycle, the denaturation time was increased to 5 min, and in the last cycle, the annealing/extension time was increased to 10 min to ensure completeness of the extension.
  • PCR reaction mixture Twenty microliters of the PCR reaction mixture was electrophoresed on a composite gel consisting of 1% agarose (Bethesda Research Laboratories) and 2% Nuseive GTG (FMC Bioproducts; Rockland, ME) . DNA was visualized by ethidium bromide staining and UV transillumination.
  • Example 6 Comparison of modified RS-PCR and conventional RT-PCR To compare the sensitivity of modified RS-PCR to conventional RT-PCR, serial ten-fold dilutions of Xenopus insulin RNA (10 7 to 10 4 molecules) were amplified using either modified RS-PCR (RT primer d 17 -t 30 ; PCR primers u 30 and t 30 ) (FIGURE 6, panel a, lanes 1-5) , or conventional RT-PCR (RT primer d 30 ; PCR primers u 30 and d 30 ) (FIGURE 6, panel a, lanes 6-
  • Modified RS-PCR was equally sensitive to conventional RT-PCR when beginning with an RNA template.
  • conventional RT-PCR resulted, as expected, in a strong signal (FIGURE 6, panel b, lane 11)
  • the modified RS-PCR method virtually ignored theDNA template (FIGURE 6, panel b, lane 10) .
  • larger amounts of DNA i.e., > 10 8 copies
  • a faint signal was detected with RS-PCR [Shuldiner et al.. Gene 91, 139 (1990)].
  • PCR was performed with two 377 bp Xenopus insulin RS- PCR products (approximately 10 8 copies) that were identical to each other except each contained a different unique tag (sequence t 30 or t' 30 (Table I and FIGURE 7) .

Abstract

Procédés de détection d'une séquence d'A.R.N. consistant à marquer la séquence avec une séquence nucléotide aléatoire unique durant la transcription inverse. La séquence nucléotide unique est alors utilisée pour amplifier sélectivement la séquence d'A.D.N. résultante. La présente invention réduit le nombre de faux positifs obtenus en raison de la contamination del'A.D.N.
PCT/US1991/002211 1990-04-05 1991-04-04 Reaction en chaine de polymerase specifique de reproduction d'a.r.n. modifiee WO1991015601A1 (fr)

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Cited By (21)

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US5399491A (en) * 1989-07-11 1995-03-21 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5554516A (en) * 1992-05-06 1996-09-10 Gen-Probe Incorporated Nucleic acid sequence amplification method, composition and kit
WO1997029211A1 (fr) * 1996-02-09 1997-08-14 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services VISUALISATION PAR RESTRICTION (RD-PCR) DES ARNm EXPRIMES DE MANIERE DIFFERENTIELLE
US5766849A (en) * 1989-07-11 1998-06-16 Gen-Probe Incorporated Methods of amplifying nucleic acids using promoter-containing primer sequence
WO1999020798A1 (fr) * 1997-10-23 1999-04-29 Exact Laboratories, Inc. Procedes de detection de contamination dans le diagnostic moleculaire utilisant la reaction pcr
WO2000008208A2 (fr) * 1998-08-05 2000-02-17 Medical Research Council Transcription inverse, amplification, et leurs amorces
WO2001006004A2 (fr) * 1999-07-19 2001-01-25 Cambridge University Technical Services Ltd. Procede d'amplification de sequences d'acide nucleique peu abondantes et moyens mis en oeuvre pour ce procede
EP1171634A1 (fr) * 1999-04-08 2002-01-16 Oasis Biosciences, Inc. Paires d'amorces d'amplification et de sequen age et leur emploi
US6818404B2 (en) 1997-10-23 2004-11-16 Exact Sciences Corporation Methods for detecting hypermethylated nucleic acid in heterogeneous biological samples
EP1508624A1 (fr) * 2003-08-22 2005-02-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Méthode de quantification pour des virus intégrés
WO2005113803A1 (fr) * 2004-05-19 2005-12-01 Amplion Limited Detection de la contamination d'amplicons lors de la pcr presentant deux temperatures de recuit differentes
US7009041B1 (en) 1989-07-11 2006-03-07 Gen-Probe Incorporated Oligonucleotides for nucleic acid amplification and for the detection of Mycobacterium tuberculosis
WO2007146154A1 (fr) * 2006-06-06 2007-12-21 Gen-Probe Incorporated Oligonucléotides marqués et leur utilisation pour des procédés d'amplification d'acides nucléiques
US7790877B2 (en) 1997-10-02 2010-09-07 Gen-Probe, Incorporated Antisense oligonucleotides with increased RNase sensitivity
US7833716B2 (en) 2006-06-06 2010-11-16 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US8153772B2 (en) 1997-10-02 2012-04-10 Gen-Probe Incorporated Oligonucleotide probes and primers comprising universal bases for diagnostic purposes
US8198027B2 (en) 2006-12-21 2012-06-12 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
US8512955B2 (en) 2009-07-01 2013-08-20 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
EP2746405A1 (fr) * 2012-12-23 2014-06-25 HS Diagnomics GmbH Procédés et ensembles d'amorces de séquençage de PCR à haut rendement
EP3042962A1 (fr) * 2015-01-07 2016-07-13 Universitätsklinikum Erlangen Détection de transcrit sens/antisens assisté par étiquette
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