Nucleic acid clonal amplification and sequencing methods, systems, and kits
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.
1. 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 attached to a surface at a 5′ end of said second strand, wherein said second strand has having-sequence complementarity with said first strand, wherein said first strand comprises a template region that is attached at a 3′ end of said template region to an adapter comprising, in a 5′ to 3′ direction, 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 an additional adapter located at a 5′ end 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 and retaining hybridization between said second sequence and said fourth sequence;
(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 additional adapter of said second strand, wherein said third strand is complementary to at least a portion of said third sequence and said fourth sequence, and wherein said primer extension separates said second sequence from said fourth sequence.
2. The method of claim 1 , 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.
3. The method of claim 2 , 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.
4. The method of claim 1 , wherein said additional adapter is immobilized to a support.
5. The method of claim 1 , wherein said first strand, at a 5′ end, further comprises a third adapter comprising, in a 3′ to 5′ direction an additional third sequence and an additional fourth sequence adjacent to said additional third sequence, wherein said additional third sequence and said additional fourth sequence are respectively hybridized to an additional first sequence and an additional second sequence at a 3′ end of said second strand, wherein said additional first sequence hybridized to said additional third sequence has said first melting point and said additional second sequence hybridized to said additional fourth sequence has said second melting point.
6. The method of claim 1 , 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 another first strand and another second strand attached to said surface at a 5′ end of said another second strand having sequence complementarity with said another first strand, wherein said another first strand comprises another template region that is attached at a 3′ end of said another template region to another adapter comprising said first sequence and said second sequence adjacent to said first sequence, wherein said first sequence and said second sequence are respectively hybridized to said third sequence and said fourth sequence of said another second strand, 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:
(e) 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 (e) is performed simultaneously with (b)-(d).
7. The method of claim 1 , wherein said adapter further comprises a fifth sequence, wherein said second sequence and said fifth sequence are disposed 3′ of 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 are disposed 5′ of said third sequence.
8. 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 a 3′ end of said first nucleic acid molecule, wherein said adapter, in a 5′ to 3′ direction, 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.
9. The method of claim 8 , further comprising attaching an additional adapter to a 5′ end of said first nucleic acid molecule.
10. The method of claim 8 , 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.
11. The method of claim 8 , wherein (b) further comprises attaching an additional adapter to a 5′ end of said first nucleic acid molecule comprising, in a 3′ to 5′ direction, an additional third sequence and an additional fourth sequence, and wherein in (c) said second nucleic acid molecule of said double-stranded nucleic acid molecule further comprises, in a 5′ to 3′ direction, an additional first sequence and an additional second sequence adjacent to said additional first sequence, wherein said additional first sequence and said additional second sequence are respectively hybridized to said additional third sequence and said additional fourth sequence, wherein said additional first sequence hybridized to said additional third sequence has said first melting point and said additional second sequence hybridized to said additional fourth sequence has said second melting point-using said adapter as a primer to conduct a primer extension reaction to yield said second nucleic acid molecule hybridized the said first nucleic acid molecule.
12. 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 at a 3′ end of said template region to a first adapter comprising, in a 5′ to 3′ direction, 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 at a 5′ end of said sequence complementary to said template region to a second adapter comprising, in a 3′ to 5′ direction, 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 and retaining hybridization between said second sequence and said fourth sequence; (ii) further denature said second sequence from said fourth sequence and hybridize said primer molecule to said second sequence of said first strand; and (iii) generate a copy of said second strand.
13. The method of claim 12 , 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 another first strand and another second strand having sequence complementarity with said another first strand, wherein said another first strand comprises a template region that is attached to another first adapter comprising in a 5′ to 3′ direction, said first sequence and said second sequence adjacent to said first sequence, and said another second strand comprises a sequence complementary to said another template region that is attached to another second adapter comprising said third sequence and said 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; 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.
14. The method of claim 12 , 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.
15. The method of claim 1 , wherein conditions sufficient to partially denature comprise exposure of said double-stranded nucleic acid molecule or a portion thereof to a chemical denaturant.
16. The method of claim 1 , wherein said first melting point is at least 1° C. lower than said second melting point.
17. The method of claim 1 , 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.
18. The method of claim 1 , wherein conditions sufficient to partially denature comprise heating said double-stranded nucleic acid molecule to partially denature said double-stranded nucleic acid molecule.
19. The method of claim 1 , wherein said adapter is immobilized to a support, and wherein said support comprises a bead.
20. The method of claim 1 , wherein each of said first sequence and said third sequence comprises at least 5 nucleotides.