Proportional amplification of nucleic acids
AssigneeAffymetrix, Inc.
InventorsYanxiang Cao, David J. Lockhart, Rui Mei, Xing Su
Life of this patent
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Abstract
The proportional amplification of nucleic acids can increase the amount of nucleic acids while preserving the relative abundance of the individual nucleic acid species, or portions thereof, in the original sample. A proportionally amplified nucleic acid preparation may be analyzed in a gene expression monitoring system, preferably involving a nucleic acid probe array.
Claims22
A method for amplification of a population of nucleic acids comprising a population of RNA to reduce amplification bias, said method comprising performing the following steps in the following order:
- synthesizing a single-stranded DNA population from said population of RNA;
- fragmenting said single-stranded DNA population to produce a fragmented single-stranded DNA population;
- synthesizing double-stranded DNA from said fragmented single-stranded DNA population;
- and
- producing multiple copies of sense RNA from said double-stranded DNA.
A method for the amplification of nucleic acid wherein amplification bias is reduced, said method comprising performing the following steps in the following order:
- contacting mRNA having a poly (A) tail with a first primer comprising poly d(T);
- generating first strand cDNA from the mRNA by extending the first primer by reverse transcriptase;
- fragmenting the first strand cDNA to produce single-stranded cDNA fragments;
- adding a poly d(A) tail to the 3′ end of the single-stranded cDNA fragments;
- contacting the single-stranded cDNA fragments with a second primer comprising a T7 promoter sequence and poly d(T);
- forming a double stranded DNA by extending the second primer;
- forming a double stranded DNA promoter region by adding the appropriate reagents;
- and
- producing multiple copies of RNA from the double stranded DNA strand comprising the promoter region.
Dependencies are inferred from the claim text.