Multiplex amplification of polynucleotides
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 kit for carrying out multiplex amplifications, comprising:
a plurality of amplification primer sets suitable for carrying out a multiplex amplification and one or more single-stranded oligonucleotide probes which are complementary to all or a part of an amplified target sequence packed in a single container, and
a plurality of reaction vessels, each of which comprises a single set of amplification primers suitable for amplifying a sequence of interest.
2. The kit of claim 1 , further comprising a DNA polymerase enzyme, a reverse transcriptase enzyme and/or mixtures of nucleoside triphosphates suitable for extension of the primers via template-dependent DNA synthesis.
3. The kit of claim 1 , further comprising reagents for carrying out downstream assays.
4. The kit of claim 3 , wherein said reagents for carrying out downstream assays are oligonucleotide probes for single nucleotide polymorphism detection or analysis, oligonucleotide microarrays and/or tailed primers for universal amplification, detection and/or purification.
5. The kit of claim 1 , further comprising reagents suitable for carrying out a plurality of singleplex quantitative or real-time amplification reactions.
6. The kit of claim 5 , wherein said reagents suitable for carrying out a plurality of singleplex quantitative or real time amplification reactions are a set of quantitative or real-time amplification primers, an oligonucleotide probe labeled with a labeling system suitable for monitoring the quantitative real-time amplification reaction, a DNA polymerase at a concentration suitable for singleplex amplification reaction and/or mixtures of deoxy nucleoside triphosphates suitable for template dependent DNA synthesis.
7. The kit of claim 1 , wherein the number of said plurality of amplification primer sets is at least 100.
8. The kit of claim 2 , wherein said DNA polymerase enzyme is Taq or Tth.
9. The kit of claim 1 , wherein said single set of amplification primers in each of said plurality of reaction vessels can independently of one another be the same or different as one of said plurality of amplification primer sets.
10. The kit of claim 1 , wherein said plurality of reaction vessels is a multi-well plate.
11. The kit of claim 1 , further comprising a 5′-exonuclease probe.
12. The kit of claim 1 , wherein each primer in said plurality of amplification primer sets is provided at a concentration of 30 nM to 100 nM.
13. The kit of claim 2 , wherein said reverse transcriptase is AMV reverse transcriptase, MuLV, or Tth reverse transcriptase.
14. A multi-part multiplex amplification composition comprising:
a first part comprising a plurality of first-stage amplification primer sets suitable for carrying out a multiplex amplification and one or more first-stage single-stranded oligonucleotide probes which are complementary to all or a part of an amplified target sequence; and
a separate, second part comprising a plurality of second-stage amplification primer sets, each of which is suitable for amplifying a different sequence of interest, and a plurality of second-stage single stranded oligonucleotide probes, each of which corresponds to one of the second-stage amplification primer sets and each of which is complementary to all or part of at least one of said sequences of interest,
wherein the second part is divided into multiple separate aliquots, each comprising a second-stage amplification primer set and its corresponding second-stage single stranded oligonucleotide probe.
15. The multi-part multiplex amplification composition of claim 14 , wherein the first-stage amplification primer sets and the second-stage amplification primer sets are the same.
16. The multi-part multiplex amplification composition of claim 14 , wherein the second part is contained within aliquots of a multi-well plate.
17. The multi-part multiplex amplification composition of claim 14 , wherein all the first-stage single-stranded oligonucleotide probes are configured to provide the same signaling.
18. The multi-part multiplex amplification composition of claim 14 , wherein the first-stage amplification primer sets and the second-stage amplification primer sets have melting temperatures that are approximately the same.