METHODS AND COMPOSITIONS FOR ENRICHMENT OF AMPLIFICATION PRODUCTS
In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods.
1 .- 105 . (canceled)
106 . A method of nucleic acid processing, comprising:
(a) providing a reaction mixture comprising: (i) a polymerase, (ii) a circular polynucleotide;
and (iii) a plurality of primers; and
(b) in said reaction mixture, subjecting said circular polynucleotide to a plurality of cycles of amplification using at least said polymerase and said plurality of primers to generate a plurality of amplification products, wherein an amplification product of said plurality of amplification products comprises more than one copy of a sequence of said circular polynucleotide, and wherein a cycle of said plurality of cycles of amplification comprises:
annealing a primer of said plurality of primers to said circular polynucleotide at an annealing temperature;
with said primer annealed to said circular polynucleotide, performing a primer elongation reaction at an elongation temperature to yield said amplification product coupled to said circular polynucleotide; and
subjecting said amplification product coupled to said circular polynucleotide to denaturation at a denaturation temperature.
107 . The method of claim 106 , wherein said circular polynucleotide is a circularized cell-free nucleic acid.
108 . The method of claim 106 , wherein said circular polynucleotide is a circularized ribonucleic acid (RNA).
109 . The method of claim 108 , wherein said circularized RNA comprises messenger RNA (mRNA) or micro-RNA (miRNA).
110 . The method of claim 106 , wherein said circular polynucleotide is a circularized deoxyribonucleic acid (DNA).
111 . The method of claim 106 , wherein said circular polynucleotide is derived from a biological sample of a subject.
112 . The method of claim 111 , wherein said biological sample is a cell-free biological sample.
113 . The method of claim 111 , wherein said biological sample is serum, plasma, blood, perspiration, saliva, urine, stool, semen, mucosal excretions, spinal fluid, amniotic fluid, or lymph fluid.
114 . The method of claim 106 , wherein said circular polynucleotide is single stranded.
115 . The method of claim 106 , wherein said denaturation temperature is different from said annealing temperature and said elongation temperature.
116 . The method of claim 115 , wherein said denaturation temperature is between about 75° C. and about 95° C.; said annealing temperature is between about 45° C. and about 65° C.; and said elongation temperature is between about 65° C. and about 75° C.
117 . The method of claim 106 , wherein said annealing temperature is different than said elongation temperature.
118 . The method of claim 106 , wherein said denaturation temperature is greater than said annealing temperature.
119 . The method of claim 106 , wherein said denaturation temperature is greater than said elongation temperature.
120 . The method of claim 106 , wherein said denaturation temperature is greater than said elongation temperature; and said elongation temperature is greater than said annealing temperature.
121 . The method of claim 106 , wherein said primer comprises a random sequence.
122 . The method of claim 106 , wherein said primer comprises a gene specific sequence.
123 . The method of claim 106 , wherein said primer comprises a barcode sequence.
124 . The method of claim 106 , wherein said primer comprises an adaptor sequence.
125 . The method of claim 106 , wherein said polymerase has strand displacement activity.