AMPLIFICATION AND ANALYSIS OF WHOLE GENOME AND WHOLE TRANSCRIPTOME LIBRARIES GENERATED BY A DNA POLYMERIZATION PROCESS
First Claim
1. A method of preparing a plurality of nucleic acid molecules having known constant region at each end, comprising:
- a) obtaining a sample comprising nucleic acid molecules;
b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers are composed of a constant region and a variable region, the constant region being positioned 5′
to the variable region, wherein primers in the population comprise non-complementary and non-self-complementary nucleotides selected from guanines and adenines;
cytosines and thymidines/uridines;
adenines and cytosines;
or guanines and thymidines/uridines in a relative proportion effective to render the polynucleotides substantially incapable of at least one of the following;
self-hybridization;
self-priming;
hybridization to another polynucleotide in the plurality;
orinitiation of a polymerization reaction in the plurality; and
c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end.
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Abstract
The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.
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Citations
59 Claims
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1. A method of preparing a plurality of nucleic acid molecules having known constant region at each end, comprising:
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a) obtaining a sample comprising nucleic acid molecules; b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers are composed of a constant region and a variable region, the constant region being positioned 5′
to the variable region, wherein primers in the population comprise non-complementary and non-self-complementary nucleotides selected from guanines and adenines;
cytosines and thymidines/uridines;
adenines and cytosines;
or guanines and thymidines/uridines in a relative proportion effective to render the polynucleotides substantially incapable of at least one of the following;self-hybridization; self-priming; hybridization to another polynucleotide in the plurality;
orinitiation of a polymerization reaction in the plurality; and c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 14, 15, 16, 17, 31, 38, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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10-13. -13. (canceled)
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18-30. -30. (canceled)
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32-37. -37. (canceled)
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39-47. -47. (canceled)
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49. (canceled)
Specification