Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
First Claim
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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 are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′
to 3′
orientation a constant region and a variable region, wherein the constant region sequence has a known sequence that is constant among a plurality of primers of the population and the variable region sequence is degenerate among said plurality of primers of the population, and further wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under the conditions employed in step c); and
c) subjecting said nucleic acid molecule/primer mixture to a polymerase under isothermal 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
51 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 are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′
to 3′
orientation a constant region and a variable region, wherein the constant region sequence has a known sequence that is constant among a plurality of primers of the population and the variable region sequence is degenerate among said plurality of primers of the population, and further wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under the conditions employed in step c); andc) subjecting said nucleic acid molecule/primer mixture to a polymerase under isothermal 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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Specification