Method of detecting gene expression and/or of preventing such expression in cells

• US 5,989,873 A
• Filed: 09/11/1997
• Issued: 11/23/1999
• Est. Priority Date: 09/24/1996
• Status: Expired due to Term

• Current Assignee: Eric Schloss, Vinayagamoorthy, Thuraiayah
• Original Assignee: Eric Schloss, Vinayagamoorthy, Thuraiayah
• Inventors: Vinayagamoorthy, Thuraiayah, Schloss, Eric
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/929,302
• Time in Patent Office: 803 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 435/91.21, 435/91.5, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/24.5, 514/44
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6851 Quantitative amplification

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.

Claims

1. A method of amplifying a nucleotide sequence complementary to an mRNA template derived from genomic DNA, which comprises:

providing a sample mixture containing an mRNA template and corresponding genomic DNA, said genomic DNA including at least two exons separated by at least one intron,

introducing intron-blockers into said mixture, said intron-blockers comprising a sequence of intron-specific deoxyribonucleotides modified to prevent nucleotide extension in conditions promoting polymerase chain reaction, and

carrying out reverse transcription polymerase chain reaction on said mRNA template, to amplify said cDNA, in the presence of a polymerase and a primer pair promoting amplification of cDNA derived from said mRNA template.

2. A method according to claim 1 wherein said polymerase does not have 3'"'"'-5'"'"' exonuclease activity, and wherein said intron-specific deoxyribonucleotides have been modified to prevent chain extension by a method selected from the group consisting of:

a) replacement of an --OH group at a 3^rd position of a last nucleotide at the 3'"'"'-end of the deoxyribo oligonucleotide, resulting in non-availability of the hydroxyl group for phosphodiester fromation;

b) removal or molecular change of a purine or pyrimidine base molecule of a last nucleotide at the 3'"'"'-end of the deoxyribo oligonucleotide, resulting in a lack of annealing of the nucleotide at this end; and

c) providing a non-phosphodiester bond between a penultimate and a final nucleotide at the 3'"'"'-end of the deoxyribooligonucleotide, creating stoichiometric constraints for purine or pyrimidine base pairing.

3. A method according to claim 1 wherein said polymerase has 3'"'"'-5'"'"' exonuclease activity, and wherein said intron-specific deoxyribooligonucleotides have been modified to prevent chain extension by a method selected from the group consisting of:

a) replacement of an --OH group at a 3^rd position of a last nucleotide at the 3'"'"'-end of the deoxyribo oligonucleotide, resulting non-availability of the hydroxyl group for phosphodiester formation; and

b) removal or molecular change of a purine or pyrimidine base molecule of a last nucleotide at the 3'"'"'-end of the deoxytribooligonucleotide, and formation of a non-phosphodiester bond between a penultimate and a final nucleotide a the 3'"'"'-end of the deoxyribo oligonucleotide, preventing 3'"'"'-5'"'"' exonuclease activity.

4. A method of detecting a specific mRNA derived from genomic DNA, which comprises:

providing a sample mixture potentially containing an mRNA template and corresponding genomic DNA, said genomic DNA including at least two exons separated by at least one intron,

introducing intron-blockers into said mixture, said intron-blockers comprising a sequence of intron-specific deoxyribooligonucleotides modified to prevent nucleotide extension in conditions promoting polymerase chain reaction,

carrying out reverse transcription polymerase chain reaction to amplify said cDNA in the presence of a polymerase and a primer pair promoting amplification of cDNA derived from said mRNA, and

detecting amplified cDNA as a confirmation of mRNA in said sample mixture.

5. A method according to claim 4 employing deoxyribooligonucleotides incorporating detectable labels as said primers and detecting said amplified DNA by detecting said labels.

6. A method of distinguishing fetal cells from maternal cells in a sample taken from a pregnant female, comprising:

providing a sample potentially containing fetal cells or maternal cells;

preparing said sample for reverse transcriptase polymerase chain reaction;

introducing intron-blockers into said prepared sample, said intron-blockers comprising oligonucleotides modified to prevent nucleotide extension in conditions promoting polymerase chain reaction, and having sequences complementary to intron regions of an alpha feto protein gene;

carrying out reverse transcription polymerase chain reaction on said mRNA template in the presence of a primer pair promoting amplification of cDNA derived from said mRNA template transcribed from said alpha feto protein gene; and

detecting amplified cDNA as a confirmation of a presence of said alpha feto protein gene as confirmation that said cells are fetal cells and hence distinguish the fetal cells from maternal cells.

7. A method of detecting viral infection in tissue transplants, which comprises detecting expression of mRNA characteristic of viral DNA in a sample of transplant tissue, by:

treating said sample to prepare said sample for reverse transcriptase polymerase chain reaction,

introducing intron-blockers into said treated sample, said intron-blockers comprising a sequence of intron-specific deoxyribooligonucleotides modified to prevent nucleotide extension in conditions promoting polymerase chain reaction, said deoxyribo oligonucleotides annealing to intron regions within said cellular genomic DNA, and

carrying out reverse transcription polymerase chain reaction, in the presence of a polymerase and a primer pair promoting amplification of cDNA derived from said viral mRNA.