METHODS FOR MULTIPLEX AMPLIFICATION
Methods for multiplex amplification of target nucleic acid sequences are provided.
1 . A method of amplifying at least two different target nucleic acid sequences in a sample comprising:
forming a plurality of different reaction compositions that each comprise a portion of the sample and at least two primer sets, wherein at least two of the primer sets are specific for a set of at least two different target nucleic acid sequences that are predicted to be present in similar abundance in the sample, wherein at least two of the primer sets of each of the plurality of different reaction compositions are different from primer sets in other reaction compositions of the plurality of different reaction compositions, such that different sets of target nucleic acid sequences are amplified in different reaction compositions during the at least one amplification reaction; and
subjecting the plurality of different reaction compositions to at least one amplification reaction to amplify the sets of different target nucleic acid sequences.
2 . The method of claim 1 , wherein at least two of the different sets of target nucleic acid sequences comprise different target nucleic acid sequences that are in similar abundance in the sample.
3 . The method of claim 1 , wherein at least one of the at least two different sets of target nucleic acid sequences in one of the reaction compositions is at least 1000 times more abundant than at least one of the at least two different sets of target nucleic acid sequences of another different reaction composition.
4 . The method of claim 1 , wherein one reaction composition comprises at least one of the at least two different sets of target nucleic acid sequences at a copy number of 1 to 10 and another reaction composition comprises at least one of the at least two different sets of target nucleic acid sequences at a copy number of 1000 to 10,000.
5 . The method of claim 1 , wherein at least one of the different sets of target nucleic acid sequences comprises target nucleic acid sequences that are in similar abundance in the sample than other sets of target nucleic acid sequences.
6 . The method of claim 1 , wherein the forming of at least one of the reaction compositions further comprises combining with the sample and the at least two primer sets (a) polymerase at a minimum concentration of at least 0.015 U/μL, (b) dNTP's at a minimum concentration of at least 2 mM, and (c) magnesium at a minimum concentration of at least 1.5 mM.
7 . The method of claim 6 , wherein the forming of at least one of the reaction compositions comprises combining with the sample and the at least two primer sets at least one of the polymerase, the dNTP's, and the magnesium in a concentration greater than the minimum concentration.
8 . The method of claim 1 , wherein the amplification reaction is performed in a high-throughput assay system.
9 . The method of claim 8 , wherein the high-throughput assay system is at least one assay system selected from an Applied Biosystems plate-reader system, the ABI 7900 Micro Fluidic Card system, microfluidic systems that exploit the use of TaqMan probes, the Invader® system, the OpenArray™ system, systems including integrated fluidic circuits (Fluidigm), and other card reader microfluidic systems.
10 . The method of claim 1 , wherein amplification products of at least two different target nucleic acid sequences that are predicted to be present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is within five to ten-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
11 . The method of claim 1 , wherein amplification products of at least two different target nucleic acid sequences that are predicted to be present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is 10 to 100-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
12 . The method of claim 1 , wherein amplification products of at least two different target nucleic acid sequences that are predicted to be present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is 100 to 1000-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
13 . A method of amplifying at least two different target nucleic acid sequences in a sample comprising:
forming a plurality of different reaction compositions that each comprise a portion of the sample and at least two primer sets, wherein at least two of the primer sets are specific for a set of at least two different target nucleic acid sequences that are present in similar abundance in the sample, wherein at least two of the primer sets of each of the plurality of different reaction compositions are different from primer sets in other reaction compositions of the plurality of different reaction compositions, such that different sets of target nucleic acid sequences are amplified in different reaction compositions during the at least one amplification reaction; and
subjecting the plurality of different reaction compositions to at least one amplification reaction to amplify the sets of different target nucleic acid sequences.
14 . The method of claim 13 , wherein at least two of the different sets of target nucleic acid sequences comprise different target nucleic acid sequences that are in similar abundance in the sample.
15 . The method of claim 13 , wherein at least one of the at least two different sets of target nucleic acid sequences in one of the reaction compositions is at least 1000 times more abundant than at least one of the at least two different sets of target nucleic acid sequences of another different reaction composition.
16 . The method of claim 13 , wherein one reaction composition comprises at least one of the at least two different sets of target nucleic acid sequences at a copy number of 1 to 10 and another reaction composition comprises at least one of the at least two different sets of target nucleic acid sequences at a copy number of 1000 to 10,000.
17 . The method of claim 13 , wherein at least one of the different sets of target nucleic acid sequences comprises target nucleic acid sequences that are in similar abundance in the sample than other sets of target nucleic acid sequences.
18 . The method of claim 13 , wherein the forming of at least one of the reaction compositions further comprises combining with the sample and the at least two primer sets (a) polymerase at a minimum concentration of at least 0.015 U/μL, (b) dNTP's at a minimum concentration of at least 2 mM, and (c) magnesium at a minimum concentration of at least 1.5 mM.
19 . The method of claim 18 , wherein the forming of at least one of the reaction compositions comprises combining with the sample and the at least two primer sets at least one of the polymerase, the dNTP's, and the magnesium in a concentration greater than the minimum concentration.
20 . The method of claim 13 , wherein the amplification reaction is performed in a high-throughput assay system.
21 . The method of claim 20 , wherein the high-throughput assay system is selected from an Applied Biosystems plate-reader system, the ABI 7900 Micro Fluidic Card system, microfluidic systems that exploit the use of TaqMan probes, and other card reader microfluidic systems.
22 . The method of claim 13 , wherein amplification products of at least two different target nucleic acid sequences present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is within five to ten-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
23 . The method of claim 13 , wherein amplification products of at least two different target nucleic acid sequences that are present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is 10 to 100-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
24 . The method of claim 13 , wherein amplification products of at least two different target nucleic acid sequences that are present in similar abundance are detected in one of the reaction compositions after at least one amplification reaction, and wherein the concentration of the amplification product of one of the at least two different target nucleic acid sequences is 100 to 1000-fold of the concentration of the amplification product of another of the at least two different target nucleic acid sequences.
25 . A method of amplifying at least two different target nucleic acid sequences in a sample comprising:
forming a plurality of different reaction compositions that each comprise a portion of the sample and at least two primer sets, wherein at least two of the primer sets are specific for a set of at least two different target nucleic acid sequences that are present at a copy number within 1000-fold of one another, wherein at least two of the primer sets of each of the plurality of different reaction compositions are different from primer sets in other reaction compositions of the plurality of different reaction compositions, such that different sets of target nucleic acid sequences are amplified in different reaction compositions during the at least one amplification reaction; and
subjecting the plurality of different reaction compositions to at least one amplification reaction to amplify the sets of different target nucleic acid sequences.