Polynucleotide sequencing

• US 20030013101A1
• Filed: 03/29/2002
• Published: 01/16/2003
• Est. Priority Date: 09/29/1999
• Status: Active Grant

• Current Assignee: Solexa Ltd. (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar
• Granted Patent: US 6,833,246 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease

Abstract

The invention relates to the sequencing of a target polynucleotide sequence, immobilized on a solid support, using the polymerase reaction to extend a suitable primer and characterizing the sequential addition of labelled bases. The present invention further relates to the presence of a polymerase enzyme that retains a 3′ to 5′ exonuclease function, which is induced to remove an incorporated labelled base after detection of incorporation. A corresponding non-labelled base may then be incorporated into the complementary strand to allow further sequence determinations to be made. Repeating the procedure allows the sequence of the complement to be identified, and thereby the target sequence.

Claims

1. A method for determining the sequence of a target polynucleotide on an array, comprising the steps of:

(i) contacting the array with one or more detectably-labelled bases A, T, G and C, under conditions that permit the polymerisation reaction to occur, to thereby incorporate a labelled base into a strand complementary to the target;

(ii) removing non-incorporated bases and detecting an incorporation event;

(iii) optionally repeating steps (i) and (ii) with one or more additional labelled bases, to determine a partial sequence;

(iv) contacting the array of step (iii) with a DNA polymerase having 3′

to 5′

exonuclease activity, under conditions whereby the polymerase cleaves the labelled base(s) and incorporates corresponding non-labelled base(s); and

(v) repeating steps (i)-(iv) sequentially, to determine the sequence.

2. A method according to claim 1, wherein the label is a fluorophore.

3. A method according to claim 1 or claim 2, wherein the labelled base is a chain terminator.

4. A method according to any preceding claim, wherein the non-labelled base comprises a removable blocking group which prevents further base incorporation from occurring, and wherein the blocking group is removed prior to repeating step (i).

5. A method according to any preceding claim, wherein the detection in step (ii) is carried out using optical means.

6. A method according to claim 5, wherein the optical means is a confocal microscope.

7. A method according to any preceding claim, wherein the polymerase of step (iv) is DNA polymerase I or III, the Klenow fragment, T4 or T7 polymerase.

8. A method according to any preceding claim, wherein step (i) is carried out using a polymerase lacking exonuclease activity.