Method of detecting gene expression and/or of preventing such ex

Details Method of detecting gene expression and/or of preventing such ex Method of detecting gene expression and/or of preventing such ex

1997-09-11

1999-11-23

Fredman, Jeffrey

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 6, 435 9121, 435 915, 536 243, 536 2431, 536 2433, 536 245, 514 44, C12P 1934, C12Q 168, C07H 2102

Patent

active

059898739

ABSTRACT:
A method of amplifying a nucleotide sequence complementary to an mRNA template derived from genomic DNA. The method involves the following steps. A sample mixture containing an mRNA template and corresponding genomic DNA is provided, the genomic DNA including at least two exons separated by at least one intron. A pair of intron-blockers are introduced into the mixture, the intron-blockers comprising a sequence of intron-specific oligonucleotides modified to prevent nucleotide extension in conditions promoting polymerase chain reaction. A primer pair promoting amplification of cDNA derived from the mRNA template is introduced into the mixture and then reverse transcription polymerase chain reaction is carried out to amplify cDNA. Detection of the cDNA is proof of the existence of mRNA in the sample, and thus proof of expression of the corresponding gene. The method avoids false positives caused by amplification of genomic DNA as well as cDNA based on an mRNA template. The invention includes a method of suppressing gene expression in vivo, which comprises exposing cells containing a gene to be suppressed, made up of exons and at least one intron, to intron-blockers having nucleotide sequences that bind to the intron to prevent gene expression.

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