METHODS FOR INCREASING MONOCLONAL NUCLEIC ACID AMPLIFICATION PRODUCTS
Disclosed herein, inter alia, are methods and compositions useful for increasing monoclonal nucleic acid amplification products on a solid support.
1 . A method of amplifying a template polynucleotide on a solid support, said method comprising:
(i) executing one or more amplification cycles thereby forming a plurality of immobilized amplification products comprising a cleavable site on the solid support, wherein each amplification cycle comprises:
a) hybridizing the template polynucleotide to a first oligonucleotide, wherein said first oligonucleotide comprises a cleavable site and is attached to the solid support, and extending the first oligonucleotide with a polymerase to generate an immobilized complement of the template polynucleotide;
b) denaturing the template polynucleotide and immobilized complement;
c) hybridizing the immobilized complement to a second oligonucleotide, wherein said second oligonucleotide comprises a cleavable site and is attached to the solid support, and extending the second oligonucleotide with a polymerase to generate an immobilized copy of the template polynucleotide;
(ii) contacting a fraction of the cleavable sites with a cleaving agent to remove a fraction of the plurality of immobilized amplification products; and
(iii) after step (ii), executing one or more amplification cycles.
2 . The method of claim 1 , further comprising repeating steps (ii) and (iii).
3 . The method of claim 1 , prior to step (i), executing one or more sparse-seed cycles, wherein each sparse-seed cycle comprises contacting the solid support with a plurality of template polynucleotides and forming a plurality of template complexes, wherein each template complex comprises a template polynucleotide hybridized to an immobilized oligonucleotide comprising a cleavable site; contacting the template complexes with a polymerase and extending the immobilized oligonucleotide to form a plurality of extended complements of templates; and removing a fraction of the extended complements of templates.
4 . The method of claim 1 , prior to step (ii), executing 2 to 20 amplification cycles.
5 . The method of claim 3 , comprising executing 2 to 8 sparse-seed cycles.
6 . The method of claim 1 , prior to step (ii), executing 2 to 5 amplification cycles.
7 . The method of claim 1 , wherein step (ii) comprises contacting about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the cleavable sites with said cleaving agent.
8 . The method of claim 1 , wherein said cleaving agent removes about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the plurality of amplification products.
9 . The method of claim 1 , wherein after step (ii), executing 2 to 50 amplification cycles.
10 . The method of claim 1 , wherein step (ii) further comprises incubating said fraction of cleavable sites with said cleaving agent for about 5 seconds to about 30 minutes.
11 . The method of claim 1 , wherein said 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 specifically recognized by a cleaving agent.
12 . The method of claim 1 , wherein denaturing comprises contacting said template polynucleotide with a denaturant, wherein said denaturant is a buffered solution comprising betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide (NMO), or a mixture thereof.
13 . The method of claim 1 , wherein the template polynucleotide comprises genomic DNA, complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), or noncoding RNA (ncRNA).
14 . The method of claim 1 , wherein the template polynucleotide comprises genomic DNA.
15 . The method of claim 1 , wherein the cleaving agent is a programmable endonuclease.
16 . The method of claim 15 , wherein the programmable endonuclease further comprises a guide oligonucleotide.
17 . The method of claim 16 , wherein the programmable endonuclease is a TtAgo enzyme.
16 . The method of claim 1 , further comprising sequencing one or more of the immobilized amplification products.
19 . The method of claim 18 , wherein sequencing comprises sequencing by synthesis, sequencing by binding, sequencing by ligation, or pyrosequencing.
20 . The method of claim 18 , wherein sequencing comprises sequencing by synthesis.