Identification of differentially expressed genes

• US 5,700,644 A
• Filed: 06/07/1995
• Issued: 12/23/1997
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Ariazi, Eric A., Gould, Michael N.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/480,597
• Time in Patent Office: 930 Days
• Field of Search: 435/6, 435/91.2, 435/172.3, 435/320.1, 435/91.51, 435/91.5
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection

Abstract

Methods are disclosed to identify differentially expressed genes. In one aspect, one uses subtractive hybridization to enrich for candidate genes, followed by a PCR amplified radiolabeled display of cDNA products of the hybridization. Specially modified primer binding regions are used that can be achieved either through use of trimming plasmids or non-symmetric display plasmids.

Claims

1. A method for identifying differentially expressed genes in experimental cells relative to control cells, comprising:

forming a first cDNA library from RNA that has been expressed by the experimental cells and a second cDNA library from RNA that has been expressed by the control cells, wherein individual members of the cDNA libraries are comprised of a 5'"'"' linker, a cDNA, and a 3'"'"' linker, wherein all members of said first cDNA library have the same 5'"'"' linker and 3'"'"' linker, all members of said second cDNA library have the same 5'"'"' linker and 3'"'"' linker, and within each library the 5'"'"' linker is different than the 3'"'"' linker;

removing from the libraries at least a portion of the cDNA common to both the first and second libraries by subtractive hybridization to provide enriched cDNA coding for differentially expressed genes that are present in said libraries;

wherein for each library individual members of the enriched cDNA derived from that library comprise a 5'"'"' DNA PCR (polymerase chain reaction) primer binding region and a 3'"'"' DNA PCR primer binding region said 5'"'"' and 3'"'"' DNA PCR primer binding regions comprising said 5'"'"' linker and said 3'"'"' linker, respectively, and up to no more than two nucleotides of cDNA adjacent to the respective linkers;

thereafter amplifying the enriched cDNA by PCR;

displaying the amplified DNA on a chromatography gel to separate different DNA; and

thereafter analyzing the gel to identify differentially expressed genes.

2. The method of claim 1, wherein labelled nucleotides are incorporated into the amplified, enriched cDNA during the amplification step.

3. The method of claim 1, wherein the subtractive hybridization step separately enriches for cDNA differentially expressed in each library, and thereafter enriched cDNA hybridization products from each library are separately so amplified and separately so displayed, and the displays are compared to each other to identify chromatography positions having different amounts of amplified DNA.

4. A method for identifying differentially expressed genes in experimental cells relative to control cells, comprising:

creating two separate cDNA libraries that are derived from the experimental cells and control cells respectively, wherein individual members of the cDNA libraries are comprised of a 5'"'"' PCR (polymerase chain reaction) primer binding region, a cDNA, and a 3'"'"' PCR primer binding region, with the 5'"'"' PCR primer binding region within a library being the same for the members of that library, the 3'"'"' PCR primer binding region within a library being the same for the members of that library, and within each library the 5'"'"' and 3'"'"' primer binding regions are different from each other in that library, with said primer binding regions each being made up of nucleotides that are not part of the cDNA sequence and up to no more than two nucleotides of the cDNA sequence adjacent thereto;

thereafter separately amplifying each cDNA library using polymerase chain reaction;

thereafter displaying the amplified DNA derived from each library in separate chromatography gel lanes; and

thereafter comparing DNA positions in a first such lane with those in a second such lane to select for cDNAs having in lane positions different amounts of amplified DNA.

5. A method for identifying differentially expressed genes in experimental cells relative to control cells, comprising:

forming a first cDNA library from RNA that has been expressed by the experimental cells and a second cDNA library from RNA that has been expressed by the control cells;

removing from both of the libraries at least a portion of the cDNA common to both the first and second libraries by subtractive hybridization to provide enriched cDNA coding for differentially expressed genes that are present in the libraries;

separately inserting the enriched cDNA derived from each library into trimming plasmids and for both libraries trimming the enriched cDNA from at least one end such that the resulting trimmed cDNA from both libraries has 5'"'"' and 3'"'"' DNA PCR (polymerase chain reaction) primer binding regions at least partially 5'"'"' and at least partially 3'"'"' respectively of cDNA that codes for cell mRNA, wherein DNA PCR primer 5'"'"' binding regions and 3'"'"' binding regions for both libraries are different from each other within a trimmed cDNA library;

thereafter separately amplifying the enriched, trimmed cDNA from both libraries using polymerase chain reaction;

displaying the amplified DNA on a chromatography gel to separate different DNA coding for different differentially expressed DNA;

and

analyzing the gel to identify chromatography positions having different amounts of amplified DNA, and to thereby identify differentially expressed genes.

6. The method of claim 5, wherein labelled nucleotides are incorporated into the amplified, enriched cDNA during the amplification step.