METHODS AND COMPOSITIONS FOR AMPLIFYING POLYNUCLEOTIDES
Disclosed herein, inter alia, are methods for increasing monoclonal nucleic acid amplification products on a solid support.
1 . A method of amplifying a polynucleotide on a solid support comprising a plurality of immobilized primers, said method comprising:
hybridizing a second platform primer binding sequence of a first immobilized polynucleotide to a second immobilized primer; wherein the first immobilized polynucleotide comprises a first platform primer sequence immobilized to a solid support, a template sequence, and said second platform primer binding sequence;
hybridizing a third platform primer binding sequence of a second immobilized polynucleotide to a third immobilized primer comprising a cleavable site; wherein the second immobilized polynucleotide comprises the first platform primer sequence, a template sequence, and said third platform primer binding sequence;
extending the second immobilized primer with a polymerase to form a first amplification product and extending the third immobilized primer with a polymerase to form a second amplification product comprising the cleavable site;
cleaving the cleavable site and removing the second amplification product; and
amplifying the first amplification product and the first immobilized polynucleotide.
2 . A method of forming a first immobilized polynucleotide and a second immobilized polynucleotide on a solid support, said method comprising:
contacting a solid support with a first polynucleotide and a second polynucleotide, wherein the solid support comprises a population of first platform primers, a population of second platform primers, and a population of third platform primers, wherein each third platform primer comprises a cleavable site and wherein each of said first platform primers, said second platform primers and said third platform primers are immobilized to said solid support;
hybridizing a first platform primer binding sequence of the first polynucleotide to one of said first platform primers, wherein the first polynucleotide comprises said first platform primer binding sequence, a template sequence, and a second platform primer sequence;
hybridizing a first platform primer binding sequence of the second polynucleotide to one of said first platform primers, wherein the second polynucleotide comprises said first platform primer binding sequence, a template sequence, and a third platform primer sequence;
extending the first platform primer with a polymerase to form the first immobilized polynucleotide comprising the first platform primer sequence, a complement of the template sequence, and a second platform primer binding sequence;
extending the second platform primer with a polymerase to form the second immobilized polynucleotide comprising the first platform primer sequence, a complement of the template sequence, and a third platform primer binding sequence.
3 . The method of claim 2 , further comprising hybridizing the first immobilized polynucleotide to one of said second platform primers and extending the second platform primer to form a first amplification product, and hybridizing the second immobilized polynucleotide to one of said third platform primers and extending the third platform primer to form a second amplification product comprising the cleavable site.
4 . The method of claim 3 , further comprising cleaving the cleavable site and removing the second amplification product from said solid support.
5 . The method of claim 3 , further comprising amplifying the first amplification product and the second amplification product.
6 . The method of claim 1 , wherein amplifying comprises 1 to 100 bridge-PCR amplification cycles.
7 . The method of claim 1 , wherein amplifying results in higher ratio of first immobilized polynucleotide and first amplification product relative to the second immobilized polynucleotide and second amplification product.
8 . The method of claim 1 , wherein the cleavable site comprises a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent.
9 . The method of claim 1 , wherein cleaving the cleavable site comprises contacting said cleavable site with a cleaving agent.
10 . The method of claim 9 , wherein the cleaving agent is sodium periodate, RNase, formamidopyrimidine DNA glycosylase (Fpg), endonuclease, uracil DNA glycosylase (UDG), TCEP, THPP, sodium dithionite (Na 2 S 2 O 4 ), hydrazine (N 2 H 4 ), Pd(0), or ultraviolet radiation.
11 . The method of claim 1 , wherein amplifying comprises 1 to 100 cycles of solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), or template walking amplification.
12 . The method of claim 6 , wherein amplifying comprises 1 to 100 thermal bridge polymerase chain reaction (t-bPCR) amplification, chemical bridge polymerase chain reaction (c-bPCR) amplification or chemical-thermal bridge polymerase chain reaction (cT-bPCR) amplification).
13 . A solid support comprising a plurality of amplification sites, wherein each amplification site comprises a population of first platform primers, a population of second platform primers, and a population of third platform primers, wherein
each of the third platform primers comprise a cleavable site,
each of said populations have a different platform primer binding sequence relative to each population, and
each of said different populations have a common platform primer binding sequence within each population.
14 . The solid support of claim 13 wherein the solid support is selected from a flow cell, bead, chip, capillary, plate, membrane, wafer, comb, pin, nanoparticle, multi-well container, or unpatterned solid support.
15 . The solid support of claim 13 wherein the solid support further comprises a polymer, photoresist or hydrogel layer.
16 . The solid support of any of claim 13 , wherein the solid support further comprises a plurality of immobilized oligonucleotides.
17 . A kit comprising the solid support of claim 13 .
18 . The kit of claim 17 , further comprising a first oligonucleotide comprising a first platform primer binding sequence, a second oligonucleotide comprising a second platform primer binding sequence, and a third oligonucleotide comprising a third platform primer binding sequence.
19 . The kit of claim 17 , further comprising a polymerase and a plurality of deoxynucleotides (dNTPs).
20 . The kit of claim 18 , wherein the first oligonucleotide further comprises a first sequencing primer binding sequence and optionally an index sequence, the second oligonucleotide further comprises a second sequencing primer binding sequence and optionally an index sequence, and third oligonucleotide further comprises a second sequencing primer binding sequence and an optionally an index sequence.