Methods Of Producing And Sequencing Modified Polynucleotides

• US 20100298551A1
• Filed: 12/07/2009
• Published: 11/25/2010
• Est. Priority Date: 06/28/2005
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Douglas R., McKernan, Kevin J.
• Application Number:
  US12/632,730
• Publication Number:
  US 20100298551A1
• Time in Patent Office: Days
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

Abstract

The present invention encompasses methods for producing a modified polynucleotide sequence that comprises a (e.g., one or more) phosphorothiolate linkage, methods for determining a polynucleotide sequence comprising a (e.g., one or more) phosphorothiolate linkage, and methods for separating forward and reverse extension products that comprise a (e.g., one or more) phosphorothiolate linkage. The invention also encompasses kits for producing and/or determining the sequence of a modified polynucleotide that comprises a (e.g., one or more) phosphorothiolate linkage.

Claims

1-94. -94. (canceled)

95. A method of separating one or more forward extension products from one or more reverse extension products comprising:

a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein at least one primer comprises a first tag;

b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages;

c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced;

d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and

e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer,

thereby separating one or more forward extension products from one or more reverse extension products.

96. The method of claim 95, wherein the one or more fragments that comprise a first primer and the one or more fragments that comprise a second primer are separated using the first tag.

97. A method of separating one or more forward extension products from one or more reverse extension products comprising:

a) annealing a first primer and a second primer to a template polynucleotide sequence comprising a sense nucleotide strand and an antisense nucleotide strand, wherein the first primer anneals to the sense strand and the second primer anneals to the antisense strand and wherein the first primer and the second primer each comprise a tag, wherein the tag on the first primer is distinct from the tag on the second primer;

b) extending the first primer and the second primer in the presence of one or more nucleoside triphosphates wherein at least one of the nucleoside triphosphates is modified, thereby producing at least one forward extension product and at least one reverse extension product, wherein the at least one forward extension product and the at least one reverse extension product comprises a modified nucleotide sequence having one or more phosphorothiolate linkages;

c) cleaving the one or more phosphorothiolate linkages in the at least one forward extension product and the at least one reverse extension product under conditions in which a plurality of fragments are produced;

d) identifying from among the fragments produced in c), one or more fragments that comprise a first primer and one or more fragments that comprise a second primer; and

e) separating the one or more fragments that comprise a first primer from the one or more fragments that comprise a second primer,

thereby separating one or more forward extension products from one or more reverse extension products.