COMPOSITIONS AND METHODS FOR THE AMPLIFICATION OF NUCLEIC ACIDS

• US 20150360193A1
• Filed: 02/21/2013
• Published: 12/17/2015
• Est. Priority Date: 07/26/2012
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Gunderson, Kevin L., Steemers, Frank J., Kaper, Fiona, Pokholok, Dmitry K.
• Granted Patent: US 10,870,099 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

The present disclosure relates to systems and methods for the amplification of nucleic acids, including, but not limited to, the amplification of nucleic acid libraries and whole genome amplification.

Claims

1. A method of creating a nucleic acid library from a nucleic acid sample, said method comprising:

a) providing a set of amplification primers to a nucleic acid sample, said set comprising

a plurality of random primers; and

a plurality of locus specific primers configured to amplify a plurality of predetermined regions of said nucleic acid library;

wherein said random primers are in greater abundance compared to said locus specific primers; and

b) amplifying said nucleic acid sample using said set of amplification primers thereby creating a nucleic acid library.

2. The method of claim 1, wherein the set of amplification primers is a mixture of primers.

3. The method of claim 1, wherein said random primers are from 5 to 18 nucleotides in length.

4. The method of claim 3, wherein said random primers are 9 nucleotides in length.

5. The method of claim 1, wherein a plurality of said locus specific primers are of equal length to said random primers.

6. The method of claim 1, wherein a plurality of said locus specific primers are shorter than said random primers.

7. The method of claim 1, wherein a plurality of said locus specific primers are longer than said random primers.

8. The method of claim 1, wherein said nucleic acid sample comprises a genomic DNA.

11. The method of claim 1, wherein said random amplification primers are selected from the group consisting of an AT-rich set of random amplification primers;

a set of random amplification primers comprising AT-rich 5′

tails;

a set of variable-length random amplification primers, wherein each primer comprises a random 3′

portion and a degenerate 5′

tail, said degenerate 5′

tail proportional in length to the A/T content of the random 3′

portion of the primer;

a set of Tm-normalized amplification primers, wherein each primer of said set comprises one or more base analogues that normalize the Tm of each primer to the Tm of other primers in said set of primers;

a set of random amplification primers, wherein each primer comprises a random 3′

portion and a constant 5′

priming portion;

a set of random amplification primers, wherein each primer comprises a random 3′

portion and a constant 5′

priming portion, and wherein said random 3′

portion comprises RNA;

a set of random amplification primers, wherein each primer comprises a random 3′

portion and a constant 5′

priming portion, and wherein said random 3′

portion comprises at least one non-natural base selected from the group consisting of 2-thio-dT and 2-amino-dA; and

any combination of the foregoing sets of primers.

9-10. -10. (canceled)

12-101. -101. (canceled)

102. A method of creating a nucleic acid library from a genomic nucleic acid sample comprising:

a) providing a set of amplification primers to a genomic nucleic acid sample wherein said set of primers comprises

a first plurality of random sequence primers, and

a second plurality of species specific sequence primers configured to amplify defined genomic regions in said nucleic acid sample, wherein said second plurality of primers is in equal or greater abundance compared to said plurality of random primers, and

b) amplifying said genomic nucleic acid sample using said set of amplification primers thereby creating a nucleic acid library.

108. The method of claim 102, wherein said defined regions in a genomic nucleic acid sample comprise one or more of non-repetitive regions and highly represented regions of a genomic nucleic acid sample.

109. The method of claim 102, wherein the sequences of said plurality of species specific primers are distributed essentially evenly across the genomic nucleic acid such that hybridization of said species specific primers to said defined genomic nucleic acid regions is unbiased.

112. The method of claim 102, wherein said second plurality of primers is present at equal abundance with said first plurality of primers.

113. The method of claim 102, wherein said second plurality of primers is present at greater abundance than said first plurality of primers.

114. The method of claim 102, wherein said species specific sequence primers comprise sequences of at least 3000 defined genomic regions.

103-107. -107. (canceled)

110-111. -111. (canceled)

115. A method for reducing overamplification of a region of a genome comprising

a) providing a set of amplification primers to a genomic nucleic acid sample wherein said set of primers comprises a first plurality of random sequence primers, and a second plurality of species specific sequence primers configured to block amplification of defined genomic regions in said nucleic acid sample, and

b) amplifying said genomic nucleic acid sample using said set of amplification primers thereby creating a nucleic acid library.

116. The method of claim 115, wherein said species specific sequence primers comprise a 3′

blocking group.

117. The method of claim 115, wherein said species specific sequence primers lack a 3′

OH.

118. The method of claim 115, wherein said species specific sequence primers comprise sequence specific to one or more repetitive regions of a genome.

119. The method of claim 115, wherein said species specific sequence primers comprise sequences that are specific to one or more regions of a genome which are typically overamplified.