Composition for nucleic acid sequencing

• US 7,416,844 B2
• Filed: 11/21/2005
• Issued: 08/26/2008
• Est. Priority Date: 05/19/1999
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Primary Examiner(s): Sisson; Bradley L
• Application Number:
  US11/285,422
• Publication Number:
  US 20060078937A1
• Time in Patent Office: 1,009 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 435/69.1, 435/174, 536/23.1, 536/24.31, 536/24.33, 536/25.32, 436/94, 436/800, 436/805
• US Class Current: 435/6.1
• 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 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Claims

1. A composition comprising:

(a) polymerizing enzyme;

(b) a nucleotide monomer; and

(c) the polymerizing enzyme comprising a first detectable-label bound covalently to a site on the polymerizing enzyme, the nucleotide monomer comprising a second detectable-label, wherein at least one of the labels has a fluorescence signal that undergoes a change before, during and/or after each of a sequence of nucleotide monomer incorporations due to an energy transfer interaction between the first label and the second label.

2. The composition of claim 1, wherein the polymerizing enzyme is a polymerase or reverse transcriptase.

3. The composition of claim 2, wherein the polymerase is selected from the group of Taq DNA polymerase, T7 DNA polymerase, SEQUENASE (DNA polymerase), and the Klenow fragment from E. coli DNA polymerase.

4. The composition of claim 3, wherein the polymerase comprises Taq DNA Polymerase having a label attached to an amino acid of the Taq DNA polymerase that is less than 60 Å

from a nucleotide that would be incorporated by said polymerase.

5. The composition of claim 2, wherein the reverse transcriptase is an HIV reverse transcriptase.

6. A composition comprising:

(a) a polymerizing enzyme;

(b) a deoxynucleotide triphosphate (dNTP); and

(c) the polymerizing enzyme comprising a first detectable-label bound covalently to a site on the polymerizing enzyme, and the deoxynucleotide triphosphate comprising a second detectable-label bound covalently thereto, wherein at least one of the labels has a fluorescence signal that undergoes a change before, during and/or after each of a sequence of dNTP incorporations due to an energy transfer interaction between the first detectable-label and the second detectable-label.

7. The composition of claim 6, wherein the polymerizing enzyme is a polymerase or reverse transcriptase.

8. The composition of claim 7, wherein the polymerase is selected from the group consisting of Taq DNA polymerase, T7 DNA polymerase, SEQUENASE (DNA polymerase), and the Klenow fragment from E. coli DNA polymerase.

9. The composition of claim 8, wherein the polymerase comprises Taq DNA Polymerase having a label attached to an amino acid of the Taq DNA Polymerase that is less than 60 Å

from a nucleotide that would be incorporated by said polymerase.

10. The composition of claim 7, wherein the reverse transcriptase is an HIV reverse transcriptase.