# US 2021/0230669 A1 · Nucleic acid clonal amplification and sequencing methods, systems, and kits > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0230669 A1 - Document ID: US20210230669A1 - Status: Published application - Kind: A1 - Application number: 17/150,659 - Filed: 2021-01-15 - Published: 2021-07-29 - Earliest claimed: 2018-07-19 - Assignees: Ultima Genomics, Inc. - Applicants: Ultima Genomics, Inc. - Inventors: Abizar LAKDAWALLA; Florian OBERSTRASS; Chandan SHEE - Web page: https://patentgrep.com/patent/US20210230669A1 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6855: Ligating adaptors - C12Q 1/6858: Allele-specific amplification - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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. ## Claims ### Claim at position 1 -80. (canceled) ### Claim 81 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. ### Claim 82 (depends on 81) The method of claim 81, further comprising (e) bringing an additional primer molecule having sequence complementarity with said first strand in contact with said first strand under conditions sufficient to permit said additional primer molecule to hybridize to said first strand, wherein said additional primer molecule hybridizes to said second sequence. ### Claim 83 (depends on 82) The method of claim 82, further comprising (f) subjecting said first strand comprising said additional primer molecule hybridized thereto to a primer extension reaction under conditions sufficient to generate a fourth strand hybridized to at least a portion of said first strand, thereby generating a double-stranded nucleic acid molecule comprising said first strand and said fourth strand. ### Claim 84 (depends on 81) The method of claim 81, wherein said second sequence is part of an additional adapter attached to said second strand, wherein said additional adapter is immobilized to a support. ### Claim 85 (depends on 81) The method of claim 81, wherein, subsequent to subjecting said double-stranded nucleic acid molecule to said conditions in (b), said first sequence is separated from said third sequence and said second sequence is hybridized to said fourth sequence, and wherein said primer extension reaction in (d) separates said second sequence from said forth sequence. ### Claim 86 (depends on 81) The method of claim 81, wherein said nucleic acid sample comprises a plurality of double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules comprises 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, wherein said plurality of double-stranded nucleic acid molecules comprises at least 100 double-stranded nucleic acid molecules, and wherein said plurality of double-stranded nucleic acid molecules comprise one or more different template regions; and further comprising: (g) repeating (b)-(d) for each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules of said nucleic acid sample; and (f) wherein (g) is performed simultaneously with (b)-(d). ### Claim 87 (depends on 81) The method of claim 81, wherein said adapter further comprises a fifth sequence, wherein said second sequence and said fifth sequence flank said first sequence, wherein said second strand of said double-stranded nucleic acid molecule further comprise a sixth sequence hybridized to said fifth sequence, and wherein said fourth sequence and said sixth sequence flank said third sequence. ### Claim 88 A method for processing a nucleic acid sample, comprising: (a) providing said nucleic acid sample comprising a first nucleic acid molecule comprising a single strand; (b) attaching an adapter to an end of said first nucleic acid molecule, wherein said adapter comprises a first sequence and a second sequence; and (c) using said adapter to generate a double-stranded nucleic acid molecule comprising a second nucleic acid molecule that is complementary to said first nucleic acid molecule, wherein said double-stranded nucleic acid molecule comprises a third sequence hybridized to said first sequence and a fourth sequence hybridized to said second 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. ### Claim 89 (depends on 88) The method of claim 88, further comprising attaching an additional adapter to another end of said first nucleic acid molecule. ### Claim 90 (depends on 88) The method of claim 88, wherein (b) comprises ligating said adapter to said first nucleic acid molecule, and wherein (c) comprises hybridizing a primer to said adapter and using said primer to perform a primer extension reaction to yield said second nucleic acid molecule hybridized to said first nucleic acid molecule. ### Claim 91 (depends on 88) The method of claim 88, wherein (b) comprises hybridizing said adapter to said first nucleic acid molecule, and wherein in (c) comprises using said adapter as a primer to conduct a primer extension reaction to yield said second nucleic acid molecule hybridized to said first nucleic acid molecule. ### Claim 92 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. ### Claim 93 (depends on 92) The method of claim 92, wherein said nucleic acid sample comprises a plurality of double-stranded nucleic acid molecules immobilized to said support, wherein each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules comprises 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 a first adapter comprising a first sequence and a second sequence adjacent to said first sequence, and 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 further comprising: (c) repeating (b) for each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules of said nucleic acid sample. ### Claim 94 (depends on 92) The method of claim 92, wherein said nucleic acid sample comprises a plurality of double-stranded nucleic acid molecules immobilized to said support, wherein each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules comprises a first strand hybridized to a second strand, wherein said first strand comprises a first sequence and a second sequence hybridized to a respective third sequence and fourth sequence, and wherein said second sequence is disposed closer to said support than said first sequence; and further comprising: (c) repeating (b) for each double-stranded nucleic acid molecule of said plurality of double-stranded nucleic acid molecules of said nucleic acid sample. ### Claim 95 (depends on 81) The method of claim 81, wherein conditions sufficient to partially denature comprise exposure of said double-stranded nucleic acid molecule or a portion thereof to a chemical denaturant. ### Claim 96 (depends on 81) The method of claim 81, wherein said first melting point is at least 1° C. lower than said second melting point. ### Claim 97 (depends on 81) The method of claim 81, wherein conditions sufficient to partially denature comprise heating said double-stranded nucleic acid molecule to a temperature higher than said first melting point and lower than said second melting point. ### Claim 98 (depends on 81) The method of claim 81, wherein conditions sufficient to partially denature comprise heating said double-stranded nucleic acid molecule to partially denature said double-stranded nucleic acid molecule. ### Claim 99 (depends on 81) The method of claim 81, wherein said adapter is immobilized to a support, wherein said support comprises a bead. ### Claim 100 (depends on 81) The method of claim 81, wherein said first sequence and said third sequence each comprise at least 5 bases.