Polynucleotide sequencing
A polynucleotide sequencing method includes a wash step that employs a composition including a polymerase. The composition may also include a plurality of nucleotides. The composition may be configured to prevent the polymerase from incorporating one of the plurality of nucleotides into a copy polynucleotide strand. The composition may be substantially free of Mg 2+ .
1 . A polynucleotide sequencing method comprising:
(a) incubating an incorporation composition comprising a first polymerase and a plurality of blocked, labeled nucleotides with a template polynucleotide strand such that the first polymerase incorporates one of the plurality of blocked, labeled nucleotides into a copy polynucleotide strand complementary to at least a portion of the template polynucleotide strand;
(b) identifying the blocked, labeled nucleotide incorporated into the copy polynucleotide strand;
(c) removing a label and a blocking moiety from the blocked, labeled nucleotide incorporated into the copy polynucleotide strand;
(d) washing the removed label and the blocking moiety away from the copy polynucleotide strand with a first wash composition comprising a second polymerase wherein the first wash composition is substantially free of Mg.sup.2+ to inhibit the second polymerase from incorporating a nucleotide into the copy polynucleotide strand; and
(e) repeating steps (a) to (d) until at least a partial sequence of the template polynucleotide strand is determined.
2 . The method of claim 1 , wherein the first wash composition comprises a plurality of nucleotides.
3 . The method of claim 2 , wherein the plurality of nucleotides comprises blocked nucleotides.
4 . The method of claim 2 , wherein the plurality of nucleotides comprises nucleotides labeled with a detectable moiety.
5 . The method of claim 1 , wherein the template polynucleotide strand is immobilized on a solid support.
6 . The method of claim 1 , wherein the template polynucleotide is immobilized on a surface of a flow cell.
7 . A polynucleotide sequencing method comprising:
(a) incubating an incorporation composition comprising a first polymerase and a plurality of blocked, labeled nucleotides with a template polynucleotide strand such that the first polymerase incorporates one of the plurality of blocked, labeled nucleotides into a copy polynucleotide strand complementary to at least a portion of the template polynucleotide strand;
(b) identifying the blocked, labeled nucleotide incorporated into the copy polynucleotide strand;
(c) contacting the copy polynucleotide strand into which the blocked, labeled nucleotide is incorporated with a first wash composition comprising a second polymerase wherein the first wash composition is substantially free of Mg.sup.2+ to inhibit the second polymerase from incorporating a nucleotide into the copy polynucleotide strand;
(d) removing a label and a blocking moiety from the blocked, labeled nucleotide incorporated into the copy polynucleotide strand;
(e) washing the removed label and the blocking moiety away from the copy polynucleotide strand with a second wash composition; and
(f) repeating steps (a) to (e) until at least a partial sequence of the template polynucleotide strand is determined.
8 . The method of claim 7 , wherein the first wash composition comprises a plurality of nucleotides.
9 . The method of claim 8 , wherein the plurality of nucleotides comprises blocked nucleotides.
10 . The method of claim 8 , wherein the plurality of nucleotides comprises nucleotides labeled with a detectable moiety.
11 . The method of claim 7 , wherein the template polynucleotide strand is immobilized on a solid support.
12 . The method of claim 7 , wherein the template polynucleotide is immobilized on a surface of a flow cell.