Methods and compositions for manipulating nucleic acids
The present disclosure provides methods, compositions and kits as well as systems for manipulating nucleic acids, including implementing isothermal amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using a pre-seeded solid support. Provided are rapid and efficient methods for generating template nucleic acid molecules comprising specific nucleotide sequence bound to solid support. Such methods can be used, for example, in manipulating nucleic acids in preparation for analysis methods that utilize monoclonal populations of nucleic acids.
1. A method for generating a nucleic acid template comprising a specific nucleotide sequence, comprising:
(a) obtaining a population of nucleic acid molecules in which each nucleic acid molecule comprises a nucleic acid strand having a first sequence of contiguous nucleotides at the 5′ end of the molecule, a second sequence of contiguous nucleotides at the 3′ end of the molecule and a third nucleotide sequence positioned between the first and second nucleotide sequences, wherein the first nucleotide sequence and second nucleotide sequence are different from each other, and wherein the first nucleotide sequences of the nucleic acid molecules are substantially identical and the second nucleotide sequences of the nucleic acid molecules are substantially identical among the population;
(b) subjecting the population of nucleic acid molecules to a polymerase chain reaction (PCR) cycle of nucleic acid amplification in the presence of a forward primer and a reverse primer, wherein the forward primer comprises an oligonucleotide sequence substantially identical to the first nucleotide sequence and the reverse primer comprises an oligonucleotide sequence complementary to a subsequence of the 3′ end of the second nucleotide sequence and a fourth nucleotide sequence that is not complementary to the second nucleotide sequence at the 5′ end of the oligonucleotide sequence; and
(c) subjecting the products of the cycle of amplification of (b) to a PCR cycle of amplification in the presence of the forward and reverse primers to generate multiple different nucleic acid products;
wherein:
the reverse primer does not contain a nucleotide sequence complementary to the 5′ end of the second nucleotide sequence, and
for each nucleic acid strand, only one of the products comprises a sequence of nucleotides complementary to the fourth nucleotide sequence.
2. The method of claim 1 , wherein the forward primer comprises a modified nucleotide containing an attachment thereto.
3. The method of claim 2 , wherein the attachment to the modified nucleotide comprises biotin.
4. The method of claim 1 , further comprising denaturing the products of (c) to produce single-stranded products and combining the single-stranded nucleic acids of the products of (c) to a single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence under annealing conditions thereby hybridizing the product of (c) that comprises a sequence of nucleotides complementary to the fourth nucleotide sequence to the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence to generate a partially double-stranded oligonucleotide-bound product.
5. The method of claim 4 , wherein the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence is attached to a support.
6. The method of claim 5 , wherein the support is a solid support.
7. The method of claim 5 , wherein the support is a particle or bead.
8. The method of claim 4 , further comprising extending the 3′ end of the oligonucleotide that is hybridized to the product that comprises a sequence of nucleotides complementary to the fourth nucleotide sequence thereby generating a double-stranded nucleic acid by synthesizing a nucleic acid strand comprising the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence and having a nucleotide sequence that is complementary to the product to which it is hybridized.
9. The method of claim 8 , further comprising separating the strands of the double-stranded nucleic acid.
10. The method of claim 9 , wherein the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence is attached to a support at the 5′ end and wherein the nucleic acid strand comprising the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence is attached to the support at the 5′ end through the portion of the strand that is the single-stranded oligonucleotide that is substantially identical to the fourth nucleotide sequence.
11. The method of claim 10 , further comprising isolating the nucleic acid strand attached to the support by removing the support from other nucleic acids that are not bound to the support.
12. The method of claim 8 , wherein the extending is carried out using a recombinase-polymerase amplification (RPA) reaction.
13. The method of claim 12 , wherein the RPA reaction is performed by incubating an RPA reaction mixture for 2 to 5 minutes at a temperature between 35° C. and 45° C.
14. The method of claim 8 , wherein the extending is carried out using a polymerase chain reaction (PCR).
15. The method of claim 14 , wherein the extending includes performing three PCR cycles.
16. The method of claim 1 , wherein the sequence of nucleotides complementary to the fourth nucleotide sequence corresponds to a capture sequence portion, the third nucleotide sequence is a template portion, and the forward primer is modified with a linker moiety, the method further comprising:
capturing a nucleic acid product of the different nucleic acid products on a bead support having a plurality of capture primers complementary to the capture sequence portion of the nucleic acid product, the capture primers hybridizing to the capture sequence portion of the nucleic acid product;
linking the captured nucleic acid product to a magnetic bead having a second linker moiety to form a bead assembly, the second linker moiety attaching to the first linker moiety; and
loading the bead assembly into a well of a device for nucleic acid analysis using a magnetic field, wherein the device is a sequencing device.
17. The method of claim 16 , further comprising extending the capture primer complementary to the nucleic acid product to form a sequence target nucleic acid attached to the bead support.
18. The method of claim 17 , further comprising denaturing the nucleic acid product and the sequence target nucleic acid to release the magnetic bead from the bead support.
19. The method of claim 18 , wherein denaturing includes enzymatic denaturing or denaturing in the presence of an ionic solution.
20. The method of claim 18 , further comprising amplifying the sequence target nucleic acid to form a population of sequence target nucleic acids on the bead support in the well.