Method for generating single-stranded DNA molecules

• US 5,518,900 A
• Filed: 11/23/1993
• Issued: 05/21/1996
• Est. Priority Date: 01/15/1993
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Molecular Tool, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Nikiforov, Theo T., Knapp, Michael R.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/155,746
• Time in Patent Office: 910 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/183, 536/24.33, 536/25.3, 536/25.32, 536/23.1, 536/24.3, 935/1, 935/8, 935/16, 935/76, 935/77, 935/78
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method for generating single-stranded nucleic acid molecules. The molecules contain nuclease resistant modified nucleotides, such that they are resistant to 5'"'"'→3'"'"' exonucleases.

Claims

1. A method for generating a desired single-stranded nucleic acid molecule, substantially free of any nucleic acid molecule of complementary sequence, said method comprising the steps:

A) incubating a preselected nucleic acid molecule in the presence of a primer molecule;

wherein said primer molecule is capable of hybridizing to said preselected molecule, and wherein said primer molecule contains a region of at least four phosphorothioate nucleotide residues at said primer'"'"'s 5'"'"' terminus;

B) permitting template-dependent extension of said primer to thereby form said desired nucleic acid molecule; and

C) adding to said incubation a 5'"'"'→

3'"'"' exonuclease selected from the group consisting of T7 5'"'"'→

3'"'"' exonuclease and lambda 5'"'"'→

3'"'"' exonuclease, under conditions sufficient to eliminate said preselected molecule, and to thereby generate said desired single-stranded molecule substantially free of any nucleic acid molecule of complementary sequence.

2. The method of claim 1, wherein in step B, after forming said desired nucleic acid molecule, said molecule is incubated in the presence of a second primer molecule capable of hybridizing thereto, and of being extended in a template-dependent manner to thereby form a nucleic acid molecule having a sequence substantially complementary to that of said desired molecule.

3. The method of claim 1, wherein in step B, after forming said desired nucleic acid molecule, said molecule is incubated in the presence of a second primer molecule capable of hybridizing thereto, and with at least one dideoxynucleotide derivative, but in the absence of any non-terminating deoxynucleotide derivatives;

wherein said incubation is under conditions sufficient to permit the extension of said primer in a template-dependent manner.

4. The method of claim 1, wherein said primer has a length of from about 10 to about 30 nucleotides.

5. The method of claim 4, wherein said primer has a length of from about 20 to about 25 nucleotides.

6. The method of claim 1, wherein the exonuclease resistance of said region that is resistant to a 5'"'"'→

3'"'"' exonuclease is caused by a plurality of phosphorothioate nucleotide derivatives.

7. The method of claim 1, wherein said primer is detectably labelled.

8. The method of claim 7, wherein said nucleotide that confers 5'"'"'→

3'"'"' exonuclease resistance to said primer molecule is detectably labelled.

9. The method of claim 1, wherein said detectable label is selected from the group consisting of an enzyme label, a fluorescent label, a radioisotopic label, and a chemiluminescent label.

10. The method of claim 1, wherein said desired single-stranded nucleic acid molecule is detectably labelled by the incorporation of labelled nucleotides during the template-dependent extension of the primer.

11. The method of claim 10, wherein said detectable label is selected from the group consisting of an enzyme label, a fluorescent label, a radioisotopic label, and a chemiluminescent label.

12. A method for detecting a desired exonuclease resistant amplification product of a polymerase chain reaction which comprises:

A) conducting a polymerase chain reaction with two primer molecules, wherein one of said primer molecules contains a region of at least four phosphorothioate nucleotide residues at said primer'"'"'s 5'"'"' terminus;

said reaction being sufficient to form double-stranded amplification products;

B) subsequently treating said amplification products with a 5'"'"'→

3'"'"' exonuclease selected from the group consisting of T7 5'"'"'→

3'"'"' exonuclease and lambda 5'"'"'→

3'"'"' exonuclease, under conditions to degrade oligonucleotides that lack a sufficient number of phosphorothioate bonds to render said oligonucleotides resistant to said exonuclease;

C) detecting said desired amplification product of said polymerase chain reaction by permitting said product to hybridize to a complementary oligonucleotide bound to a solid support.

13. A kit, being specially adapted to contain in close compartmentalization a first container which contains a first primer, said first primer containing a region of at least four phosphorothioate nucleotide residues at said primer'"'"'s 5'"'"' terminus;

and a second container which contains a second primer lacking any phosphorothioate nucleotide derivatives, such that the two primers can be used to amplify a predetermined gene sequence.

14. The kit of claim 13 which additionally includes a 5'"'"'→

3'"'"' exonuclease selected from the group consisting of T7 5'"'"'→

3'"'"' exonuclease and lambda 5'"'"'→

3'"'"' exonuclease.