Multiplex amplification of polynucleotides
View Patent ↗The present invention provides methods, reagents and kits for carrying out a variety of assays suitable for analyzing polynucleotides or samples that include an amplification step performed in a multiplex fashion. Also provided are methods for analyzing and improving the efficiency of amplification and for carrying out gene expression analysis.
1. A method for analyzing a sample or plurality of samples for the presence of one or more polynucleotide sequences of interest, comprising:
(i) amplifying at least one of said polynucleotides of interest in the presence of:
(a) a plurality of different amplification primer pairs suitable for amplifying said polynucleotide sequences of interest; and
(b) a plurality of oligonucleotide probes, wherein each of said plurality of oligonucleotide probes is complementary to a region of a different polynucleotide sequence of interest amplified by said plurality of primer pairs and comprises a label suitable for monitoring amplification as a function of time; and
(ii) amplifying the products of said step (i) by dividing said products of step (i) into a plurality of aliquots and performing real-time polymerase chain reaction (PCR) on at least one of said aliquots in the presence of:
(a) at least one of said primer pairs used in step (i); and
(b) at least one of said oligonucleotide probes used in step (i) wherein said oligonucleotide probe in step (ii) is complementary to a region of a polynucleotide sequence of interest amplified by said primer pair in step (ii),
wherein said step (i) amplifying is carried out for less than 40 cycles.
2. The method of claim 1 in which the amplification of step (i) is further carried out in the presence of a reverse transcriptase such that the polymerase chain reaction is reverse-transcription polymerase chain reaction and wherein the one or more polynucleotide sequences is obtained from mRNA derived from the sample.
3. The method of claim 1 in which the one or more polynucleotide sequences comprise a cDNA library.
4. The method of claim 1 in which the polymerase chain reaction of step (i) is carried out for a number of cycles such that the amplification remains in the linear range.
5. The method of claim 1 in which the amplification in step (i) is achieved with a thermostable DNA polymerase.
6. The method of claim 1 in which the label is a fluorophore.
7. The method of claim 1 in which said plurality of oligonucleotide probes is selected from the group consisting of 5′-exonuclease probes, stem-loop beacon probes and stemless beacon probes.
8. The method of claim 1 in which an observed efficiency of amplification is greater than 70%.
9. The method of claim 1 in which an observed efficiency of amplification is greater than 90%.
10. The method of claim 1 in which the amplification is carried out in the presence of uracil N-glycosylase.
11. The method of claim 1 in which the amplifying of step (i) comprises fewer than fifteen PCR cycles.
12. The method of claim 1 , wherein said less than 40 cycles is a number of cycles suitable to keep said amplification reaction within the exponential or linear phase.