Method of nucleic acid sequence selection

• US 5,500,356 A
• Filed: 08/10/1993
• Issued: 03/19/1996
• Est. Priority Date: 08/10/1993
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Lin, Jhy-Jhu, Jessee, Joel A., Li, Wu-Bo, Gruber, Christian E.
• Primary Examiner(s): Fleisher, Mindy B.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/103,769
• Time in Patent Office: 952 Days
• Field of Search: 435/91.1, 435/91.2, 435/172.3, 536/24.3, 536/24.33
• US Class Current: 506/1
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Abstract

The present invention provides a method for the rapid isolation and recovery of a desired target DNA or RNA molecules from a mixture or library containing such molecules. The method involves the use of biotinylated probes and enzymatic repair-cleavage to eliminate undesired library members from a sample.

Claims

1. A method for obtaining an enriched source of a desired target nucleic acid molecule from an initial sample containing a mixture or library of single-stranded nucleic acid containing said desired molecule, wherein said method comprises the steps:

(A) incubating said initial sample containing said nucleic acid mixture or library in the presence of a biotinylated nucleic acid probe molecule, said probe molecules having a sequence complementary to a nucleotide sequence of said desired target molecule;

said incubating being under conditions sufficient to permit said probe to hybridize to said desired target molecule and to thereby generate hybridized molecules wherein said target molecule is bound to said probe;

(B) incubating a sample containing said hybridized molecules of step (A) in the presence of a biotin-binding ligand immobilized to a support;

said incubating being sufficient to permit said hybridized molecules to become bound to said biotin-binding ligand of said support;

(C) separating said bound hybridized molecules of step, (B) from Unbound nucleic acid molecules, and recovering said bound hybridized molecules from said support, and then incubating said recovered hybridized molecules under conditions sufficient to separate the strands of double-stranded nucleic acid molecules;

said incubating thereby releasing said hybridized target molecule from said biotinylated probe, and generating single-stranded desired target molecules;

(D) incubating said single-stranded desired target molecules in the presence of a nucleotide analog and a primer nucleic acid molecule complementary to a sequence of said desired target molecule;

wherein said nucleotide analog confers endonuclease resistance to nucleic acid molecules that contain said analog;

said incubating being under conditions sufficient to permit hybridization between said primer and said single-stranded desired target molecule, and further sufficient to permit the template-dependent extension of said primer to thereby generate a double-stranded desired target molecule wherein one strand of said double-stranded desired target molecule contains said incorporated nucleotide analog and is resistant to endonuclease;

(E) incubating said generated double-stranded molecules in the presence of a nuclease capable of degrading nucleic acid that lacks said incorporated endonuclease-resistant nucleotide triphosphate derivatives, but incapable of degrading nucleic acid that contains said incorporated endonuclease-resistant nucleotide triphosphate derivatives;

wherein said incubating is conducted under conditions sufficient to degrade nucleic acid that lacks said incorporated endonuclease-resistant nucleotide triphosphate derivatives to thereby form said enriched source of said desired target nucleic acid molecule.

2. The method of claim 1, wherein in step A said incubating is under conditions which minimize random hybridization.

3. The method of claim 1, wherein said desired target nucleic acid molecule is a DNA molecule.

4. The method of claim 1, wherein said desired target nucleic acid molecule is an RNA molecule.

5. The method of claim 1, wherein said desired target molecule is a circular nucleic acid molecule.

6. The method of claim 5, wherein said desired target molecule is a circular DNA molecule.

7. The method of claim 1, wherein said biotin-binding ligand is avidin or streptavidin.

8. The method of claim 7, wherein said biotin-binding ligand is avidin.

9. The method of claim 7, wherein said biotin-binding ligand is streptavidin.

10. The method of claim 1, wherein said support is a paramagnetic bead.

11. The method of claim 10, wherein said hybridized molecule bound to said paramagnetic bead is recovered by magnetic means.

12. The method of claim 1, wherein said primer molecule of step (D) is complementary to the same sequence of said desired target molecule as said probe molecule of step (A).

13. The method of claim 1, wherein said primer molecule of step (D) is complementary to a sequence of said desired target molecule that differs from the sequence of said desired target molecule that is complementary to said probe molecule of step (A).

14. The method of claim 1, wherein said single-stranded desired target molecule contains deoxyuridine, and wherein said nuclease is uracil DNA glycosylase (UDG).

15. The method of claim 1, wherein said nuclease does not cleave hemimethylated DNA.

16. The method of claim 15, wherein said nucleic acid analog is 5-methylcytidine, and wherein said nuclease that does not cleave hemimethylated DNA is HhaI.

17. The method of claim 1, wherein in step (A), said probe has a degenerate sequence.

18. The method of claim 1, wherein in step (D), said primer has a degenerate sequence.

19. The method of claim 1, wherein said method additionally includes the step of amplifying said desired target molecule by an in vitro amplification reaction.

20. The method of claim 19, wherein said in vitro amplification reaction is a polymerase chain reaction.