US 2021/0230669 A1 · NUCLEIC ACID CLONAL AMPLIFICATION AND SEQUENCING METHODS, SYSTEMS, AND KITS

Abstract

The present disclosure provides methods and systems for processing nucleic acid samples. Methods for processing a nucleic acid sample may comprise providing a double-stranded nucleic acid molecule comprising a partially denaturable region; partially denaturing the partially denaturable region of the double-stranded nucleic acid molecule, thereby generating a region comprising two single strands; and hybridizing a priming sequence to a sequence of one of the single strands. The methods described herein may facilitate amplification without the need for a multitude of complex steps or numerous reagents.

Claims21

Dependent claims

-80. (canceled)

A method for processing a nucleic acid sample, comprising:

(a) providing said nucleic acid sample comprising a double-stranded nucleic acid molecule comprising a first strand and a second strand having sequence complementarity with said first strand, wherein said first strand comprises a template region that is attached to an adapter comprising a first sequence and a second sequence adjacent to said first sequence, wherein said first sequence and said second sequence are respectively hybridized to a third sequence and a fourth sequence of said second strand, wherein said first sequence hybridized to said third sequence has a first melting point and said second sequence hybridized to said fourth sequence has a second melting point higher than said first melting point;
(b) subjecting said double-stranded nucleic acid molecule to conditions sufficient to partially denature said double-stranded nucleic acid molecule, thereby separating said first sequence of said first strand from said third sequence of said second strand;
(c) bringing a primer molecule having sequence complementarity with said third sequence of said second strand in contact with said second strand under conditions sufficient to permit said primer molecule to hybridize to said third sequence of said second strand;
and
(d) subjecting said second strand comprising said primer molecule hybridized to said third sequence of said second strand to a primer extension reaction under conditions sufficient to generate a third strand hybridized to at least a portion of said second strand.

2 more elements

A method for processing a nucleic acid sample, comprising:

(a) providing said nucleic acid sample comprising a double-stranded nucleic acid molecule immobilized to a support, wherein said double-stranded nucleic acid molecule comprises a first strand and a second strand having sequence complementarity with said first strand, wherein:
(i) said first strand comprises a template region that is attached to a first adapter comprising a first sequence and a second sequence adjacent to said first sequence, and
(ii) said second strand comprises a sequence complementary to said template region that is attached to a second adapter comprising a third sequence and a fourth sequence adjacent to said third sequence,
wherein said first sequence and said second sequence are respectively hybridized to said third sequence and said fourth sequence,
wherein said first sequence hybridized to said third sequence has a first melting point and said second sequence hybridized to said fourth sequence has a second melting point higher than said first melting point, and
wherein said support comprises a plurality of primer molecules immobilized thereto;
and
(b) performing an amplification reaction using a primer molecule of said plurality of primer molecules by subjecting said nucleic acid sample to conditions sufficient to (i) partially denature said double-stranded nucleic acid molecule, thereby separating said first sequence of said first adapter of said first strand from said third sequence of said second adapter of said second strand;
(ii) hybridize said first primer molecule to said second sequence of said first strand;
and (iii) generate a copy of said second strand.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application

17/150,659Filed 15 Jan 2021

Priority

19 Jul 2018

Published

29 Jul 2021

Term ends

Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6855
Using modified primers or templates: ligating adaptors
C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Nucleic acid clonal amplification and sequencing methods, systems, and kits

Life of this patent

Nucleic acid clonal amplification and sequencing methods, systems, and kits

Life of this patent

Abstract

Claims21