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 - 56 . (canceled)
57 . A method of simultaneously amplifying a plurality of polynucleotide sequences of interest in a single reaction comprising:
a) selecting two or more polynucleotide sequences of interest for amplification;
b) hybridizing to the polynucleotide sequences of interest a primer set, wherein each primer set contains at least one uracil nucleobase; and
c) simultaneously amplifying the polynucleotide sequences of interest by polymerase chain reaction in a single reaction to generate a plurality of amplicons.
58 . The method of claim 57 , wherein each primer set contains a forward amplification primer and a reverse amplification primer and wherein each amplification primer contains at least one uracil nucleobase.
59 . The method of claim 58 , wherein the forward and reverse amplification primers are substantially complementary to the polynucleotide sequences of interest to be amplified.
60 . The method of claim 57 , wherein the primer sets are suitable for amplifying a different polynucleotide sequence of interest.
61 . The method of claim 57 , wherein the primer set is present in the polymerase chain reaction at a concentration of about 30 to about 100 nM.
62 . The method of claim 61 , wherein substantially all amplification primers in the polymerase chain reaction are present at a concentration of 45 nM.
63 . The method of claim 57 , wherein substantially all primer sets present in the polymerase chain reaction are approximately equimolar, in the range of about 30 nM to about 100 nM.
64 . The method of claim 57 , wherein the number of primer sets present in the polymerase chain reaction is at least 100.
65 . The method of claim 57 , wherein the polynucleotide sequences of interest are treated with uracil DNA glycosylase.
66 . The method of claim 57 , wherein the plurality of amplicons are subsequently amplified with a different set of primers from those employed in the first amplification step.
67 . The method of claim 57 , wherein the polymerase chain reaction includes a thermostable DNA polymerase.
68 . A method of generating a plurality of target sequences of interest, comprising the step of: amplifying by polymerase chain reaction one or more target polynucleotides in the presence of a plurality of amplification primers suitable for amplifying the target sequences of interest, wherein substantially all the amplification primers comprise at least one uracil nucleobase.
69 . The method of claim 68 , wherein the plurality of target sequences of interest comprises at least 1000 target sequences of interest.
70 . The method of claim 68 , wherein substantially all the amplification primers comprise about 15 to about 35 nucleotides in length.
71 . The method of claim 68 , wherein substantially all the amplification primers have a melting temperature of about 60° C. to about 78° C.
72 . The method of claim 68 , in which the amplified target sequences of interest are treated with uracil N-glycosylase.
73 . The method of claim 68 , in which the amplified target sequences of interest are analyzed for single nucleotide polymorphisms, genotyping, gene mutations for genetic diagnosis or nucleic acid sequencing.
74 . A kit for amplifying multiple polynucleotide sequences of interest in a single reaction comprising, a plurality of amplification primer sets suitable for amplifying the polynucleotide sequences of interest, wherein each primer set contains at least one uracil nucleobase, a thermostable DNA polymerase and a mixture of nucleotide triphosphates suitable for extension of the amplification primers via template dependent DNA synthesis.
75 . The kit of claim 74 , further comprising uracil N-glycosylase.
76 . An amplicon generated by the method of claim 57 .