Method for simultaneous identification of differentially expressed mRNAs and measurement of relative concentrations

• US 5,459,037 A
• Filed: 11/12/1993
• Issued: 10/17/1995
• Est. Priority Date: 11/12/1993
• Status: Expired due to Fees

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Erlander, Mark G., Sutcliffe, J. Gregor
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): Carter, Philip W.
• Application Number:
  US08/152,482
• Time in Patent Office: 704 Days
• Field of Search: 435/6, 435/91.1, 435/91.3, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

An improved method for the simultaneous sequence-specific identification of mRNAs in a mRNA population allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. In general, the method comprises the formation of cDNA using anchor primers to fix a 3'"'"'-endpoint, producing cloned inserts from the cDNA in a vector containing a bacteriophage-specific promoter for subsequent RNA synthesis, generating linearized fragments of the cloned inserts, preparing cRNA, transcribing cDNA from the cRNA using a set of primers, and performing PCR using a 3'"'"'-primer whose sequence is derived from the vector and a set of 5'"'"'-primers that is derived from the primers used for transcription of cDNA from cRNA. The method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions.

Claims

1. A method for simultaneous sequence-specific identification of mRNAs in a mRNA population comprising the steps of:

(a) isolating a mRNA population;

(b) preparing double-stranded cDNAs from the mRNA population using a mixture of 12 anchor primers with the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO;

     2), wherein V is a deoxyribonucleotide selected from the group consisting of A, C, and G; and

N is a deoxyribonucleotide selected from the group consisting of A, C, G, and T, the mixture including anchor primers containing all possibilities for V and N, to produce a cDNA sample;

(c) cleaving the cDNA sample with two restriction endonucleases, a first restriction endonuclease MspI and a second restriction endonuclease NotI;

(d) inserting the cDNA sample cleaved with the first and second restriction endonucleases into a vector, the cleaved cDNA being inserted in an orientation that is antisense with respect to a T3 promoter within the vector, the vector being the plasmid pBC SK⁺ cleaved with ClaI and NotI;

(e) transforming Escherichia coli with the vector into which the cleaved cDNA has been inserted to produce cloned inserts;

(f) generating linearized fragments of the cloned inserts by digestion with at least one restriction endonuclease that is different from the first and second restriction endonucleases;

(g) generating a cRNA preparation of antisense cRNA transcripts by incubation of the linearized fragments with a T3 RNA polymerase capable of initiating transcription from the T3-specific promoter;

(h) dividing the cRNA preparation into sixteen subpools and transcribing first-strand cDNA from each subpool, using a thermostable reverse transcriptase, and one of the sixteen primers A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO;

     3), wherein N is one of the four deoxyribonucleotides A, C, G, or T, and a different primer is used in each of the subpool

(i) using the product of transcription in each of the sixteen subpools as a template for a polymerase chain reaction with the 3'"'"'-primer G-A-A-C-A-A-A-A-G-C-T-G-G-A-G-C-T-C-C-A-C-C-G-C (SEQ ID NO;

     4), and a 5'"'"'-primer selected from the group consisting of;

(1) the primer from which first-strand cDNA was made for that subpool;

(2) the primer from which the first-strand cDNA was made for that subpool extended at its 3'"'"'-terminus by an additional residue -N, where N can be any of A, C, G, or T; and

(3) the primer used for the synthesis of first-strand cDNA for that subpool extended at its 3'"'"'-terminus by two additional residues -N-N, wherein N can be any of A, C, G, or T, to produce polymerase chain reaction amplified fragments; and

(j) resolving the polymerase chain reaction amplified fragments by electrophoresis to display bands representing the 3'"'"'-ends of mRNAs present in the sample.

2. A panel of primers comprising 16 different oligonucleotides each having the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO:

3), wherein N is one of the four deoxyribonucleotides A, C, G, or T.

3. A panel of primers comprising 64 different digonucleotides each having the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ ID NO:

5), wherein N is one of the four deoxyribonucleotides A, C, G, or T.

4. A panel of primers comprising 256 different oligonucleotides each having the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO:

6), wherein N is one of the four deoxyribonucleotides A, C, G, or T.