Methods and adaptors for generating specific nucleic acid populations

• US 6,060,245 A
• Filed: 09/26/1997
• Issued: 05/09/2000
• Est. Priority Date: 12/13/1996
• Status: Expired due to Fees

• Current Assignee: Stratagene California
• Original Assignee: Stratagene California
• Inventors: Mullinax, Rebecca L., Sorge, Joseph A.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/938,835
• Time in Patent Office: 956 Days
• Field of Search: 435/6, 435/91.2, 435/810, 536/24.2, 536/24.33
• US Class Current: 435/6.14
• 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 1/6855 Ligating adaptors

Abstract

The present invention relates to methods and kits for generating or analyzing nucleic acid populations or desired nucleic acid sequences based upon replication or amplification reactions. The invention comprises methods employing adaptors ligated to nucleic acids that preferentially permit replication or amplification of desired nucleic acid sequences or preferentially eliminate undesired nucleic acids from replication or amplification. The invention also comprises adaptors useful in the methods and in kits for replicating or amplifying nucleic acids. In one embodiment, the adaptors function to protect desired nucleic acids from cleavage by a restriction enzyme while other nucleic acids are cleaved. The protected, desired nucleic acids can then be preferentially replicated or amplified. Accordingly, the invention can be used for the amplification of desired nucleic acids and the effective removal of undesired nucleic acids from a population.

Claims

1. A method for preferentially amplifying one or more desired nucleic acids comprising:

preparing tester nucleic acids and driver nucleic acids from selected populations of nucleic acids;

producing adaptor;

tester nucleic acid complexes with said tester nucleic acids, wherein each adaptor;

tester nucleic acid complex comprises a tester nucleic acid and a first adaptor comprising a first adaptor sequence, wherein said first adaptor sequence comprises a priming site;

producing adaptor;

driver nucleic acid complexes with said driver nucleic acids, wherein said adaptor;

driver nucleic acid complex comprises a driver nucleic acid and a second adaptor comprising a second adaptor sequence, and wherein said first and second adaptor sequences are different;

combining said adaptor;

tester nucleic acid complexes and said adaptor;

driver nucleic acid complexes so as to produce double-stranded nucleic acids comprising double-stranded adaptor;

tester nucleic acid complexes, double-stranded adaptor;

driver nucleic acid complexes, and double-stranded adaptor;

tester/adaptor;

driver nucleic acid complexes, under conditions such that said first adaptor sequences of the adaptor;

tester nucleic acid complexes do not hybridize with said second adaptor sequences of the adaptor;

driver nucleic acid complexes;

adding a single-stranded nuclease that substantially cleaves single stranded nucleic acids; and

amplifying said double-stranded adaptor;

tester nucleic acid complexes with a reagent that selectively amplifies nucleic acids ligated to the first adaptor sequence.

2. The method of claim 1, wherein said reagent is a primer that recognizes nucleic acids included in the first adaptor sequence and does not recognize nucleic acids included in the second adaptor sequence.

3. A method for preferentially amplifying one or more desired nucleic acids comprising:

preparing tester nucleic acids and driver nucleic acids from selected populations of nucleic acids;

producing adaptor;

tester nucleic acid complexes with said tester nucleic acids, wherein each adaptor;

tester nucleic acid complex comprises a tester nucleic acid and a first adaptor, wherein said first adaptor comprises a priming site that is recognized by a specific amplification primer;

producing adaptor;

driver nucleic acid complexes with said driver nucleic acids, wherein each adaptor;

driver nucleic acid complex comprises a driver nucleic acid and a second adaptor, wherein said second adaptor lacks a nucleic acid sequence that is recognized by said specific amplification primer,

combining said adaptor;

tester nucleic acid complexes and said adaptor;

driver nucleic acid complexes so as to produce double-stranded nucleic acids comprising double-stranded adaptor;

tester nucleic acid complexes, double-stranded adaptor;

driver nucleic acid complexes, and double-stranded adaptor;

tester/adaptor;

driver nucleic acid complexes, under conditions such that said nucleic acid sequences that are recognized by said specific amplification primer of said adaptor;

tester nucleic acid complexes do not hybridize with said adaptor;

driver nucleic acid complexes;

adding a single-stranded nuclease that substantially cleaves single stranded nucleic acids; and

amplifying said double-stranded adaptor;

tester nucleic acid complexes using said specific amplification primer.

4. A kit for preferentially replicating or amplifying one or more desired nucleic acids comprising one or more selected adaptors wherein at least one selected adaptor comprises a priming site, one or more selected primers, a reagent capable of substantially preventing replication or amplification of all but single-stranded desired nucleic acid complexes, and reagents for replicating or amplifying said single-stranded desired nucleic acid complexes.