Transfer and detection of nucleic acids

• US 4,302,204 A
• Filed: 07/02/1979
• Issued: 11/24/1981
• Est. Priority Date: 07/02/1979
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Wahl, Geoffrey M., Stark, George R.
• Primary Examiner(s): Wiseman, Thomas G.
• Application Number:
  US06/054,200
• Time in Patent Office: 876 Days
• Field of Search: 435/6, 435/7, 435/172, 435/805, 23/230 B, 23/230.3, 23/230.6, 424/1, 424/2, 424/1.5, 422/56
• US Class Current: 436/501
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: Y10S 435/805 Test papers

Abstract

Improvements in the transfer and detection of separated nucleic acids, both RNA and DNA, are provided. For analysis of large DNA, the molecular weight segregated fractions of DNA are depurinated and fragmented to provide fractions having less than about 2 kb as single strands. With both RNA and DNA, the nucleic acid fractions are transferred after resolution to a chemically treated substrate and covalently affixed to the substrate. The resulting nucleotides affixed to the substrate are hybridized with labeled nucleotide probes and a volume exclusion agent, particularly a water soluble ionic polymer.

Claims

1. A process for the analysis of polynucleotides of at least ten bases which comprises:

combining a solid substrate having polynucleotides covalently affixed thereto with a hybridization solution containing labeled polynucleotides suspected of being complementary to said affixed polynucleotides and a charged polysaccharide of at least 10,000 molecular weight present in at least about 2 weight %; and

detecting the presence of labeled polynucleotides annealed to said affixed polynucleotides.

2. A method according to claim 1, wherein said charged polysaccharide is present in at least about 5 weight %.

3. A method according to claim 2, wherein said charged polysaccharide is dextran sulfate.

4. A method according to claim 1, wherein said polynucleotide is RNA.

5. A method according to claim 1, wherein said polynucleotide is DNA.

6. A method according to any of claims 4 and 5, wherein said charged polysaccharide is dextran sulfate and is present in at least about 5 weight %.

7. A method for analyzing double stranded DNA in a mixture having DNA molecules having chain lengths greater than 1 kb which comprises:

distributing said DNA mixture according to molecular weight on a polysaccharide gel by means of gel electrophoresis;

fragmenting and denaturing said DNA to provide single stranded DNA of less than about 2 kb;

transferring at least a portion of said fragmented single stranded DNA to a chemically reactive solid substrate to covalently bond said transferred DNA to said substrate to provide DNA-substrate;

combining said DNA-substrate with a hybridizing solution containing labeled single stranded DNA suspected of being complementary to the DNA of said DNA substrate; and

determining the presence of labeled DNA bound to said DNA of said DNA-substrate by means of said label.

8. A method according to claim 7, wherein said chemically reactive solid substrate is diazosubstituted paper.

9. A method according to claim 7, wherein said fragmenting and denaturing involves contacting said DNA zone with acid and base respectively.

10. A method according to any of claims 7 to 9, wherein said hybridizing solution contains a charged polymeric volume exclusion agent.

11. A method according to claim 10, wherein said agent is dextran sulfate of at least about 10,000 molecular weight present in at least about 10 weight %.

12. A method for analyzing double stranded DNA of chain length greater than about 1 kb in a mixture of DNA which comprises:

distributing said mixture in a gel according to molecular weight by means of gel electrophoresis;

fragmenting and denaturing at least a portion of said DNA by treating with acid and base successively to provide ssDNA of chain lengths less than about 2 kb;

transferring said ssDNA to diazo substituted paper to covalently bind said ssDNA to said paper to provide DNA-paper;

hybridizing radioactively labeled single stranded DNA in a hybridizing solution with said DNA paper, whereby complementary labeled ssDNA binds to said DNA paper and wherein said hybridizing solution is an aqueous solution of from about 40 to 60 volume % of a low molecular weight polar organic solvent and from about 5 to 25 weight % of dextran sulfate of from about 10,000 to 1 M molecular weight.