Making nucleic acid sequences in parallel and use
The present invention relates generally to the fields of genomics, synthetic biology and genetic engineering. More particularly, the present invention concerns the methods that enable parallel multiplex ligation and amplification on surface for making assemblies of nucleic acids of various biological applications and for analysis of biological samples such as DNA, RNA, and proteins.
1. A method for simultaneously producing two full-length duplex DNA polymers which have identical sequences on a solid surface comprising multiple sites wherein each of the two full-length duplex DNA polymers is located on a different site on the solid surface and wherein each duplex consists of a first strand and a second complementary strand each strand having a 5′ end and a 3′ end comprising:
(a) placing a first capture probe of a defined length which is identical to a portion of the 3′ end of the first strand on a first site on the solid surface;
(b) placing a second capture probe of a defined length which is identical to a portion of the 3′ end of the second strand on a second site on the solid surface;
(c) contacting the solid surface with a pool of partially double-stranded DNA oligonucleotides which comprise overlapping fragments of the full-length duplex DNA polymers to be produced;
(d) hybridizing the first and second capture probes with the pool of partially double-stranded DNA oligonucleotides such that a first and second full-length duplex DNA polymers are formed in which there are nicks and gaps in the duplexes;
(e) washing the first and second full-length duplex DNA polymers under conditions to remove DNA polymers which do not have sequences which are identical to the two full-length duplex DNA polymers; and
(f) ligating and extending the first gapped and unligated full-length duplex DNA and the second gapped and unligated full-length duplex DNA to produce two full-length duplex DNA polymers which have identical sequences wherein the first full-length duplex DNA polymer is attached to the first site through the 3′ end of the first strand and the second full-length duplex DNA polymer is attached to the second site through the 3′ end of the second strand.