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Processes for genetic manipulations using promoters

DC
  • US 5,891,636 A
  • Filed: 09/03/1997
  • Issued: 04/06/1999
  • Est. Priority Date: 09/22/1989
  • Status: Expired due to Term
First Claim
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1. A gene expression library derived from a cell or tissue sample, comprising two or more specific nucleic acid messages of various abundances, whose levels of representation relative to other messages within a population reflect the physiologic state of the sample, thereby permitting diagnosis of a disease or condition, wherein said library is prepared by the following steps:

  • (a) adding a primer complex to a population of messenger RNAs (mRNAs) from said cell or cell population, said primer complex comprising;

    (i) a primer sequence complementary to a plurality of said population of mRNAs of said cell or cell population, and(ii) a promoter sequence in antisense orientation, wherein said primer complex hybridizes to said plurality population of mRNAs;

    (b) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by(i) extending said primer complex to form a first cDNA strand, and(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation;

    (c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of the primer complex; and

    (d) quantitating specific aRNAs corresponding to specific mRNAs,wherein a plurality of said aRNAs represents an expression spectrum in said cell or cell population.

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