CLONAL AMPLIFICATION OF NUCLEIC ACID ON SOLID SURFACE WITH TEMPLATE WALKING
First Claim
Patent Images
1. A method of primer extension, comprising:
- a) hybridizing a first primer molecule (“
first forward primer”
) to a complementary primer-binding sequence (“
reverse-strand PBS”
) on a nucleic acid strand (“
reverse strand”
);
wherein at least 60% of nucleotide bases of the first forward primer are adenine, thymine or uracil or are complementary to adenine, thymine or uracil;
b) generating an extended first forward strand that is a full-length complement of the reverse strand and is hybridized thereto, by extending the first forward primer molecule using the reverse strand as template; and
c) hybridizing a second primer molecule (“
second forward primer”
) to the reverse-strand PBS where the reverse strand is also hybridized to the first forward strand.
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Abstract
Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.
80 Citations
31 Claims
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1. A method of primer extension, comprising:
-
a) hybridizing a first primer molecule (“
first forward primer”
) to a complementary primer-binding sequence (“
reverse-strand PBS”
) on a nucleic acid strand (“
reverse strand”
);
wherein at least 60% of nucleotide bases of the first forward primer are adenine, thymine or uracil or are complementary to adenine, thymine or uracil;b) generating an extended first forward strand that is a full-length complement of the reverse strand and is hybridized thereto, by extending the first forward primer molecule using the reverse strand as template; and c) hybridizing a second primer molecule (“
second forward primer”
) to the reverse-strand PBS where the reverse strand is also hybridized to the first forward strand. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17-25. -25. (canceled)
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26. A method of generating separated and immobilized clonal populations of a first template sequence (“
- template 1”
) and a second template sequence (“
template 2”
), comprising amplifying the first and second template sequence, wherein;a) both templates are in single-stranded form and are both contained within the same continuous liquid phase, where a first and second immobilization site (respectively, “
IS1” and
“
IS2”
) are in contact with said continuous liquid phase, and where IS1 and IS2 are spatially separated,b) template 1 comprises a first subsequence (“
T1-FOR”
) at its 3′
end, and a second subsequence (“
T1-REV”
) that is non-overlapping with T1-FOR and at its 5′
end,c) template 2 comprises a first subsequence (“
T2-FOR”
) at its 3′
end, and a second subsequence (“
T2-REV”
) that is non-overlapping with T2-FOR and at its 5′
end,d) IS1 comprises an immobilized primer (“
IS1 primer”
) that can hybridize to both T1-FOR and T2-FOR,e) IS2 comprises an immobilized primer (“
IS2 primer”
) that can hybridize to both T1-FOR and T2-FOR,f) the reverse complement of T1-REV cannot hybridize substantially to primers on IS1, and/or the reverse complement of T2-REV cannot hybridize substantially to primers on IS2, but can each hybridize substantially to a non-immobilized primer in the continuous liquid phase; whereby amplification results in a population of clonal amplicons of template 1 substantially attached to IS1 and not to IS2, and/or a population of clonal amplicons of template 2 substantially attached to IS2 and not to IS1. - View Dependent Claims (27, 28, 29, 30)
- template 1”
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31-103. -103. (canceled)
Specification