DNA sequencing using controlled strand displacement
This application discloses methods of producing a DNA strand for sequencing, as well as genetic constructs, libraries, and arrays using DNA strands produced according to these methods. The application also discloses methods of sequencing using the DNA strands, genetic constructs, libraries, and arrays produced. In certain aspects, DNA being sequenced includes a target sequence and at least one adaptor sequence.
1 . A method of producing a DNA strand for sequencing, comprising
a) providing a template DNA polynucleotide comprising a first target DNA sequence interposed between a first adaptor 3′ to the first target DNA sequence and a second adaptor 5′ to the first target DNA sequence, and optionally comprising a third adaptor 3′ to the first adaptor and a second target DNA sequence interposed between the first adaptor and the third adaptor, wherein the template DNA polynucleotide is immobilized on a substrate,
b) combining a first primer with the immobilized template DNA polynucleotide, and hybridizing the first primer to a first primer binding sequence in the first adaptor, wherein the first primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide;
c) extending the first primer using a first DNA polymerase to generate a second strand, wherein the second strand comprises a sequence complementary to the first target DNA sequence and a sequence complementary to at least part of the second adaptor;
d) combining a second primer with the immobilized template DNA polynucleotide, hybridizing a second primer to a second primer binding sequence, wherein the second primer binding sequence is 3′ to the first primer binding sequence, wherein the second primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide; and
e) extending the second primer using a DNA polymerase having strand-displacement activity under controlled conditions, wherein the reaction is controlled by selecting temperature, enzyme concentration, and primer concentration, and/or is terminated at a fixed time interval to generate a third strand,
wherein said controlled conditions are selected such that extending the second primer to generate the third strand partially displaces, but does not completely displace, the second strand, thereby producing a partially hybridized second strand having:
(i) a hybridized portion that is hybridized to the template DNA polynucleotide, and
(ii) an unhybridized overhang portion that contains a sequence that is complementary to the first target DNA sequence and a sequence that is complementary to at least part of the second adaptor, wherein the unhybridized portion is 5′ in the second strand to the hybridized portion.
2 . The method of claim 1 , further comprising:
f) hybridizing a sequencing oligonucleotide to the sequence in the unhybridized overhang portion of the second strand that is complementary to at least part of the second adaptor, and
g) determining at least part of the sequence that is complementary to the first target DNA sequence.
3 . The method of claim 1 , wherein the first adaptor, the second adaptor, and the third adaptor if present, have the same nucleotide sequence.
4 . The method of claim 1 , wherein said first DNA polymerase and said DNA polymerase having strand-displacement activity are the same polymerase.
5 . The method of claim 1 , wherein the second primer binding sequence, to which the second primer is hybridized, is in the first adaptor.
6 . The method of claim 1 , wherein the template DNA polynucleotide comprises the third adaptor and the second primer binding sequence is in the third adaptor.
7 . The method of claim 1 , wherein the template DNA polynucleotide comprises a DNA concatemer, and the first target DNA sequence and the second target DNA sequence have the same nucleotide sequence.
8 . The method of claim 4 , wherein the first primer and the second primer are hybridized or extended in the same reaction.
9 . The method of claim 1 , wherein the template DNA polynucleotide comprises a DNA concatemer and the first primer and the second primer have the same nucleotide sequence.
10 . The method of claim 7 , wherein step d) further comprises hybridizing a plurality of second primers to a plurality of second primer binding sequences, wherein the plurality of second primer binding sequences are in adaptor sequences of the concatemer, and wherein the plurality of second primers comprise extendable and non-extendable primers.
11 . The method of claim 1 , wherein extension of the second primer is terminated at a fixed time interval of 5 min, 10 min, 20 min, 30 min, 40 min or 60 min, and wherein extension is terminated by chemical termination and/or addition of ddNTPs.
12 . The method of claim 1 , wherein the template DNA polynucleotide is deposited on arrays, beads, wells, or droplets.
13 . The method of claim 1 , wherein the sequencing is sequencing by synthesis, pyrosequencing, or sequencing by ligation.