Multiplex nucleic acid amplification
In some embodiments, the disclosure relates generally to compositions, comprising a single reaction mixture containing a plurality of different populations of discrete supports, and a plurality of different populations of target nucleic acids. The single reaction mixture can contain a first population of beads; a second population of beads; a first population of target nucleic acids, where at least two different target nucleic acids in the first population of target nucleic acids can bind to a bead in the first population of beads; and a second population of target nucleic acids, where at least two different target nucleic acids in the second population of target nucleic acids can bind to a bead in the second population of beads. The single reaction mixture can be employed to monoclonally amplify the first target nucleic acids on the first beads, and monoclonally amplify the second target nucleic acids on the second beads.
1. A method of nucleic acid synthesis, comprising:
a) providing a single reaction mixture that provides compartmentalization in an emulsion containing a plurality of supports of a first type that include first capture primers; a plurality of supports of a second type that include second capture primers, wherein the sequences of the first and second capture primers are different, a first population of nucleic acids, wherein the first population of nucleic acids includes at least one nucleic acid of a first adapter type, a second population of nucleic acids, wherein the second population of nucleic acids includes at least one nucleic acid of a second adapter type, wherein the first and second adapter types are different, a first fusion primer having a first sequence portion identical to at least a portion of the first capture primer and a second sequence portion that binds to the first adapter type, and a second primer having a first sequence portion identical to at least a portion of the second capture primer and a second sequence portion that binds to the second adapter type;
b) forming a first amplified population of nucleic acids by amplifying one or more nucleic acids of the first population of nucleic acids, wherein the first amplified population of nucleic acids is attached to one or more supports of the first type; and
c) forming a second amplified population of nucleic acids by amplifying one or more nucleic acids from the second population of nucleic acids, wherein the second amplified population of nucleic acids is attached to one or more supports of the second type.
2. The method of claim 1 , wherein forming the first amplified population of nucleic acids and/or forming the second amplified population of nucleic acids includes amplifying a nucleic acid from the first and/or second population of nucleic acids, respectively, by PCR, isothermal or rolling circle amplification.
3. The method of claim 1 , wherein the first amplified population of nucleic acids, the second amplified population of nucleic acids or both are substantially monoclonal.
4. The method of claim 1 , wherein the emulsion includes a water-in-oil emulsion.
5. The method of claim 1 , wherein the capture primers are not labeled.
6. The method of claim 1 , wherein the single reaction mixture includes at least two, three, four, five, six, seven, eight, nine, ten, or more types of supports, and wherein each type of support includes a different capture primer.
7. The method of claim 1 , further comprising sequencing one or more nucleic acids of the first amplified population and one or more nucleic acids of the second amplified population in parallel.
8. The method of claim 7 , wherein the sequencing includes detection of one or more nucleotide incorporation byproducts.
9. The method of claim 7 , wherein the sequencing includes detecting hydrogen ions or pyrophosphate.
10. The method of claim 1 , wherein the first and second capture primers are present in about the same concentration.
11. The method of claim 1 , wherein forming the first amplified population of nucleic acids includes extending the at least one nucleic acid sequence from the first population of nucleic acids to form an extended portion complementary to the first sequence portion of the first fusion primer and hybridizing the extended portion of the first population of nucleic acids to a first capture primer on the first type of supports; and forming the second amplified population of nucleic acids includes extending the at least one nucleic acid sequence from the second population of nucleic acids to form an extended portion complementary to the second sequence portion of the second fusion primer and hybridizing the extended portion of the second population of nucleic acids to a second capture primer on the second type of supports.
12. The method of claim 1 , further including extending one or more first and second capture primers in a template-dependent fashion.
13. A method of amplifying a first nucleic acid sequence from a first population of nucleic acids and a first nucleic acid sequence from a second population of nucleic acids comprising:
(a) providing a single reaction mixture that provides compartmentalization in an emulsion containing a plurality of a first type of supports that include first capture primers, a plurality of a second type of supports that include second capture primers, wherein the first and second capture primers are different, a first fusion primer that includes a portion identical or complementary to the first capture primers, a second fusion primer that includes a portion identical or complementary to the second capture primers, a first population of nucleic acids, wherein the first population of nucleic acids includes at least one nucleic acid comprising sequence identical or complementary to a portion of a first fusion primer and a second population of nucleic acids, wherein the second population of nucleic acids includes at least one nucleic acid comprising sequence identical or complementary to a portion of a second fusion primer;
(b) forming a first amplified population of a first nucleic acid sequence from the first population of nucleic acids on the first type of supports using the first fusion primer; and
(c) forming a second amplified population of the first nucleic acid sequence from the second population of nucleic acids on the second type of supports using the second fusion primer.
14. The method of claim 13 , wherein the first amplified population, the second amplified population or both are substantially monoclonal.
15. The method of claim 13 , wherein the first amplified population of nucleic acids, the second amplified population of nucleic acids, or both, are substantially monoclonal.
16. The method of claim 13 , wherein the amplifying includes PCR, isothermal or rolling circle amplification.
17. The method of claim 13 , wherein the emulsion includes a water-in-oil emulsion.
18. The method of claim 13 , wherein the first amplified population of nucleic acids is covalently bound to one or more supports of the first plurality of supports and/or the second amplified population of nucleic acids is covalently bound to one or more supports of the second plurality of supports.
19. The method of claim 13 , wherein the single reaction mixture further includes at least two, three, four, five, six, seven, eight, nine, ten, or more types of supports, each type of support including a different type of capture sequence.
20. The method of claim 13 , further comprising sequencing one or more nucleic acids of the first amplified population and one or more nucleic acids of the second amplified population in parallel.