MULTIPLEX AMPLIFICATION OF POLYNUCLEOTIDES

• US 20130096014A1
• Filed: 10/04/2012
• Published: 04/18/2013
• Est. Priority Date: 12/04/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: ANDERSEN, Mark, RUFF, David
• Application Number:
  US13/645,264
• Publication Number:
  US 20130096014A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

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.

Claims

1-56. -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.

76. An amplicon generated by the method of claim 57.

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.