Nucleic acid molecules containing recombination sites and methods of using the same

• US 8,304,189 B2
• Filed: 12/01/2004
• Issued: 11/06/2012
• Est. Priority Date: 12/01/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chesnut, Jon, Leong, Louis
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US11/000,371
• Publication Number:
  US 20050208530A1
• Time in Patent Office: 2,897 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase
• 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 2535/125 Allele specific primer extension

Abstract

The present invention relates to the fields of biotechnology and molecular biology. In particular, the present invention relates to the construction and use of nucleic acid molecules comprising cloning sites which differ in nucleotide sequence. In particular embodiments, the present invention relates to nucleic acid molecules which contain recombination sites with different primer binding sites. These different primer binding sites may be used to sequence different ends of nucleic acid segments located between the two recombination sites.

Claims

1. A method for sequencing all or part of a nucleic acid segment, the method comprising:

(a) providing a first nucleic acid molecule comprising the nucleic acid segment flanked by a first attP recombination site and a second attP recombination site which can not recombine with each other and wherein the first and the second attP recombination sites each have a sequencing primer binding site located within 30 nucleotides of the nucleic acid segment wherein the sequencing primer binding sites encompass the IHF site of the first and the second attP site and wherein the sequencing primer binding sites differ from each other by one, two, three or four nucleotides,

(b) providing a second nucleic acid molecule comprising a first attB recombination site and a second attB recombination site which can not recombine with each other,

(c) combining the first and the second nucleic acid molecules in the presence of one or more recombinases under conditions which allow for site specific recombination in trans between,

(1) the first attP recombination site and the first attB recombination site and,

(2) the second attP recombination site and the second attB recombination site,

such that a product nucleic acid molecule is generated which contains the nucleic acid segment flanked by a first and a second attL recombination sites, wherein the product attL recombination sites comprise the unique binding sites encompassing the IHF site from the attP recombination sites,

(d) sequencing all or part of a first strand of the product nucleic acid molecules comprising the attL sites with a sequencing primer which mediates 5′

to 3′

extension from the unique sequencing primer binding site located within the first attL recombination site, and

(e) sequencing all or part of a second strand of the nucleic acid segment comprising the attL sites with a sequencing primer which mediates 5′

to 3′

extension from the sequencing primer binding site located within the second attL site specific recombination site.

2. The method of claim 1, wherein, when the sequencing primer binding sites differ from each other by two, three or four nucleotides, the nucleotides which are different are located adjacent to each other.

3. A method for sequencing all or part of a nucleic acid segment comprising:

(a) providing in a recombination reaction, a first nucleic acid comprising the nucleic acid segment flanked by att recombination sites which do not recombine with each other, wherein the att recombination sites each comprise an IHF site, a second nucleic acid molecule comprising two att recombination sites which cannot recombine with each other but can recombine with the att recombination sites in the first nucleic acid, one or more recombinases, wherein the two att recombination sites within the second nucleic acid molecule each comprise sequencing primer binding sites that encompass the IHF sites wherein the recombination reaction is effective to generate a product nucleic acid molecule comprising the two sequencing primer binding sites flanking the nucleic acid segment wherein the two sequencing primer sites are within 30 nucleotides of the nucleic acid segment and wherein the two sequencing primer binding sites differ from each other in nucleotide sequence by one, two, three, or four nucleotides and allow for sequencing of the nucleic acid segment from either end;

(b) contacting a composition comprising the product nucleic acid molecule of (a), comprising the sequencing primer binding sites, with a first sequencing primer wherein the first sequencing primer binds to both sequencing primer binding sites but mediates 5′

to 3′

extension only when bound to one of the two sequencing primer binding sites; and

(c) sequencing from the end of the nucleic acid segment comprising the bound first primer.

4. The method of claim 3, further comprising the steps of:

(d) contacting a second composition comprising the product nucleic acid molecule of (a), comprising the sequencing primer binding sites, with a second sequencing primer wherein the second sequencing primer binds to both sequencing primer binding sites but mediates 5′

to 3′

extension only from the sequencing primer binding site located at the opposite end of the nucleic acid segment from which the first sequencing primer mediates 5′

to 3′

extension; and

(e) sequencing from the end of the nucleic acid segment comprising the bound second primer.

5. The method of claim 4, wherein the first sequencing primer and the second sequencing primer are each between 15 and 45 nucleotides in length.

6. The method of claim 5, wherein the first sequencing primer and the second sequencing primer differ in nucleotide sequence at their 3′

termini.

7. The method of claim 6, wherein the first sequencing primer comprises the nucleotide sequence 5′

GTTGCAACAAATTGATGAGCAATTA 3′

(SEQ ID NO;

     1) and the second sequencing primer comprises the nucleotide sequence 5′

GTTGCAACAAATTGATGAGCAATGC 3′

(SEQ ID NO;

     2).