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. An array comprising a substrate comprising a plurality of discrete areas, wherein each of at least a subset of the plurality of discrete areas comprises a DNA complex, said DNA complex comprising
a first DNA strand that comprises a target sequence,
a second DNA strand that is complementary to the first DNA strand,
a third DNA strand that is complementary to the first DNA strand, and
a fourth DNA strand that is complementary to the second DNA strand,
wherein the first DNA strand is a single-stranded DNA concatemer immobilized on the discrete area,
wherein the second DNA strand comprises a 5-prime portion that is complementary to, and not hybridized to, the first DNA strand, and a 3-prime portion that is complementary to, and hybridized to, a second adaptor comprising a second adaptor sequence in the first DNA strand,
the third DNA strand is complementary to, and hybridized to, both a first adaptor and the target sequence in the first DNA strand,
wherein the first adaptor comprises a first adaptor sequence,
wherein the second adaptor sequence and the first adaptor sequence are the same, and
the fourth DNA strand comprises a polynucleotide comprising at least a portion of the second adaptor sequence the sequence, and
wherein said polynucleotide is hybridized to the 5-prime portion of the second DNA strand.
2. A system comprising the substrate according to claim 1 and one or more DNA polymerases.
3. The system of claim 2 , wherein at least one of the one or more DNA polymerases has strand displacement activity.
4. The system of claim 2 , wherein one or more DNA polymerases comprise Phi29.
5. A system comprising the array of claim 1 and a ligase.
6. The array of claim 1 , wherein the array comprises a mixture of extendable and non-extendable primers.
7. The array of claim 1 , wherein single-stranded DNA concatemers immobilized on different discrete areas comprise different target sequences.
8. A DNA complex comprising:
a first DNA strand,
a second DNA strand that is complementary to the first DNA strand,
a third DNA strand that is complementary to the first DNA strand, and
a fourth DNA strand that is complementary to the second DNA strand,
wherein the first DNA strand is a single-stranded DNA concatemer immobilized on a solid support,
wherein the second DNA strand comprises a 5-prime portion that is complementary to, and not hybridized to, the first DNA strand, and a 3-prime portion that is complementary to, and hybridized to, a second adaptor in the first DNA strand, wherein the second adaptor comprising a second adaptor sequence,
the third DNA strand is complementary to, and hybridized to, both a first adaptor and the target sequence in the first DNA strand,
wherein the first adaptor comprises a first adaptor sequence,
wherein the second adaptor sequence and the first adaptor sequence are the same, and
the fourth DNA strand comprises a polynucleotide comprising at least a portion of the second adaptor sequence, and
wherein said polynucleotide is hybridized to the 5-prime portion of the second DNA strand.