Quantitative multiplex PCR with high dynamic range
View Patent ↗The invention provides a method for quantitative multiplex PCR of two targets whereby one target is present in at least 100-fold molar excess over that of the other target. The method further comprises the addition of a thermostable DNA polymerase with a final concentration of at least 0.5 units/μl.
1. A method for measuring amounts of at least two mRNAs in a sample by multiplex PCR, comprising the steps of:
incubating said at least two mRNAs with a reverse transcriptase under conditions effective to produce a single-stranded cDNA from each mRNA, wherein at least one high-abundance mRNA is present in at least a 100-fold molar excess over at least one low-abundance mRNA, and wherein said mRNAs are present in copy numbers between 10 2 and 10 8 ;
amplifying said single-stranded cDNAs with a thermostable DNA polymerase at a concentration of at least 0.5 units DNA polymerase/μl and a pair of primers for each single-stranded cDNA under conditions effective to amplify said single-stranded cDNAs in a real-time polymerase chain reaction, whereby the amplification of said low-concentration mRNA is inhibited in the presence of said high-concentration mRNA by not more than 10%, relative to amplification in the absence of said high-concentration mRNA;
determining the amount of each of said amplified single-stranded cDNAs by real-time measurement of an amount of fluorescent emission, wherein the amount of fluorescent emission provides a quantitative value of the amount of said amplified single-stranded cDNA; and
determining the amount of each of said mRNAs in said sample from the amount of said cDNA derived from said mRNA.
2. The method of claim 1 , wherein said step of incubating said single-stranded cDNAs with a thermostable DNA polymerase is performed using a hot start technique.
3. The method of claim 2 , wherein said thermostable DNA polymerase is modified with a heat labile blocking group.
4. The method of claim 2 , wherein said thermostable DNA polymerase is inactivated at ambient temperatures by addition of oligonucleotide aptamers.
5. The method of claim 2 , wherein said thermostable DNA polymerase is inactivated at ambient temperatures by addition of an anti-DNA polymerase antibody, said antibody binding to said polymerase and inhibiting polymerization by said polymerase at temperatures below those of said real-time polymerase chain reaction, said antibody further being releasable from said polymerase at temperatures of said real-time polymerase chain reaction.
6. The method of claim 1 , wherein said fluorescent emission is obtained by excitation of a single-stranded nucleic acid probe labeled with a fluorescent moiety and incubated with said single-stranded cDNAs.
7. The method of claim 1 , wherein said fluorescent emission is obtained by excitation of at least one pair of FRET hybridization probes labeled with a fluorescent moiety and incubated with said single-stranded cDNAs.
8. The method of claim 1 , wherein said fluorescent emission is obtained by excitation of at least one TaqMan hybridization probe labeled with a fluorescent moiety and incubated with said single-stranded cDNAs, and said thermostable DNA polymerase has 5′–3′ exonuclease activity.
9. The method of claim 1 , wherein said fluorescent emission is obtained by excitation of at least one molecular beacon labeled with a fluorescent moiety and incubated with said single-stranded cDNAs.
10. The method of claim 1 , wherein said step of amplifying said single-stranded cDNAs is performed for less than one minute per amplification cycle.