Method for sequencing synthetic oligonucleotides containing non-phosphodiester internucleotide linkages

• US 5,403,709 A
• Filed: 10/06/1992
• Issued: 04/04/1995
• Est. Priority Date: 10/06/1992
• Status: Expired due to Fees

• Current Assignee: Avecia Biotechnology Inc (Nitto Denko Corporation)
• Original Assignee: Hybridon, Inc. (Aceragen Incorporated)
• Inventors: Agrawal, Sudhir, Tang, Jin-Yan
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US07/958,133
• Time in Patent Office: 910 Days
• Field of Search: 435/6, 435/91, 435/91.1, 436/94
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to synthetic oligonucleotides and more particularly to the determination of nucleotide sequences of synthetic oligonucleotides having non-phosphodiester internucleotide linkages. The invention provides a method for sequencing such modified synthetic oligonucleotides.

Claims

1. A method for determining the nucleotide sequence of a single-stranded sample oligonucleotide except for its 5'"'"'-terminal nucleotide, comprising the steps of:

(a) annealing the sample oligonucleotide and a single-stranded helper oligonucleotide, each having 5'"'"' and 3'"'"' ends, with a single-stranded molecular tack oligonucleotide having a 5'"'"' region complementary to the 5'"'"' end of the helper oligonucleotide and an immediately adjacent 3'"'"' region complementary to the 3'"'"' end of the sample oligonucleotide, the sequence of at least three of the 3'"'"'-terminal nucleotides of the sample oligonucleotide being known;

(b) ligating the helper oligonucleotide to the sample oligonucleotide to yield a sequencing-length oligonucleotide;

(c) annealing a labelled primer oligonucleotide to the helper oligonucleotide portion of the sequencing-length oligonucleotide, the primer oligonucleotide having a nucleotide sequence that is complementary to a portion of the helper oligonucleotide;

(d) extending the primer oligonucleotide with a polymerase in the presence of chain-extending and chain terminating nucleoside triphosphates;

(e) fractionating the extended primers according to standard procedures; and

(f) determining the nucleotide sequence of the sample oligonucleotide, except for its 5'"'"'-terminal nucleotide, from the fractionated extended primers.

2. The method according to claim 1, wherein the sample oligonucleotide has one or more non-phosphodiester internucleotide linkage at a 3'"'"' end.

3. The method according to claim 2, wherein the non-phosphodiester internucleotide linkage is a phosphorothioate linkage.

4. A method for determining the nucleotide sequence of a single-stranded sample oligonucleotide except for its 5'"'"'-terminal nucleotide comprising the steps of:

(a) annealing the sample oligonucleotide and a single-stranded helper oligonucleotide, each having 5'"'"' and 3'"'"' ends, with a single-stranded molecular tack oligonucleotide having a 5'"'"' region complementary to the 5'"'"' end of the helper oligonucleotide and an immediately adjacent 3'"'"' region complementary to the 3'"'"' end of the sample oligonucleotide, the sequencing at least three of the 3'"'"'-terminal nucleotides of the sample oligonucleotide being known;

(b) ligating the helper oligonucleotide to the sample oligonucleotide to yield a sequencing-length oligonucleotide;

(c) annealing a labelled primer oligonucleotide to the helper oligonucleotide portion of the sequencing-length oligonucleotide, the primer oligonucleotide having a nucleotide sequence that is complementary to a portion of the helper oligonucleotide;

(d) extending the primer oligonucleotide with a polymerase to yield a primer extension product;

(e) cleaving the primer extension product with appropriate Maxam and Gilbert sequencing reagents to yield cleaved primer extension products;

(f) fractionating the cleaved primer extension products according to standard procedures; and

(g) determining the nucleotide sequence of the sample oligonucleotide except for its 5'"'"'-terminal nucleotide, from the fractionated cleaved primer extension products.

5. The method according to claim 4, wherein the sample oligonucleotide has one or more non-phosphodiester internucleotide linkage at a 3'"'"' end.

6. The method according to claim 5, wherein the non-phosphodiester internucleotide linkage is a phosphorothioate linkage.