RNA fingerprinting to determine RNA population differences

• US 5,665,544 A
• Filed: 06/09/1995
• Issued: 09/09/1997
• Est. Priority Date: 05/27/1992
• Status: Expired due to Fees

• Current Assignee: Amersham International Plc (General Electric Company)
• Original Assignee: Amersham International Plc (General Electric Company)
• Inventors: Beabealashvili, Robert Shavlovich, Chenchik, Alexander Anatoljevich, Carter, Christopher John, Diachenko, Ludmila Borisovna
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/343,449
• Time in Patent Office: 823 Days
• Field of Search: 435/6, 435/5, 536/24.3, 935/77, 935/80
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

A method of producing an RNA fingerprint of a poly(A)⁺ RNA preparation. At least one oligonucleotide primer and a supply of nucleotides of which one is a terminator nucleotide, are added to the poly(A)⁺ RNA preparation or a single-stranded cDNA preparation derived from it. cDNA products are synthesized from the RNA, or DNA products from the cDNA and the products are separated to produce an RNA fingerprint.

Claims

1. A method of producing a fingerprint of a eukaryotic poly(A)+ RNA preparation comprising;

adding to the poly(A)+ RNA preparation or to a single-stranded cDNA preparation derived therefrom at least one oligonucleotide primer and four different nucleotides, wherein three of the nucleotides permit elongation and one of the nucleotides is a chain terminator;

synthesizing cDNA products from said RNA preparation or DNA products from said single-stranded cDNA preparation; and

separating said cDNA products or DNA products to produce a fingerprint.

2. The method according to claim 1, wherein the oligonucleotide primer(s) is(are) 8 to 10 nucleotides in length.

3. The method according to claim 1, wherein the number of primers and nucleotide sequence of the primers are chosen to provide a sufficiently detailed fingerprint to allow the fingerprints obtained from different poly(A)+ RNA or cDNA preparations to be distinguished from each other.

4. The method according to claim 1, wherein the chain terminating nucleotide is any modified nucleotide triphosphate possessing chain terminating properties when used with any DNA polymerase.

5. The method according to claim 1, wherein the chain terminating nucleotide is dideoxyuridine triphosphate.

6. The method according to claim 1, wherein the chain terminating nucleotide is dideoxyuridine triphosphate containing either a biotin or a fluorescein group at the 5-position of the pyrimidine ring.

7. The method according to claim 1, wherein the separating of the cDNA products or DNA products is carried out by electrophoresis.

8. The method according to claim 1, wherein the primer(s) is(are) labeled with a detectable label.

9. The method according to claim 1, wherein the chain terminating nucleotide is labeled with a detectable label.

10. The method according to claim 1, wherein said poly(A)+ RNA preparation is isolated from a tissue selected from the group consisting of normal human tissues, fetal tissues, cirrhotic human liver tissues, human cancerous tissues, uterine myoma tissue, and mouse testicular teratocarcinoma cells.

11. A method for comparing the poly(A)+ RNA in two or more cell populations comprising,

producing a fingerprint of a poly(A)+ preparation for each cell population by the method according to claim 1, and

visually or otherwise comparing the separated cDNA products or DNA products in the respective fingerprints.

12. The method according to claim 11, wherein one or more of the separated cDNA products or DNA products are further compared by isolating and sequencing the products.