Methods for manipulating biomolecules
In some embodiments, the present teachings provide compositions, systems, methods and kits for generating a population of nucleic acid fragments. In some embodiments, nucleic acids can be fragmented enzymatically. For example, methods for generating a population of nucleic acid fragments can include a nucleic acid nicking reaction. In one embodiment, the methods can include a nick translation reaction. A nicking reaction can introduce nicks at random positions on either strand of a double-stranded nucleic acid. A nick translation reaction can move the position of nicks to a new position so that the new positions of two of the nicks are aligned to create a double-stranded break. In some embodiments, methods for generating a population of nucleic acid fragments can include joining at least one end of a fragmented nucleic acid to one or more oligonucleotide adaptors.
1 . A method for preparing a library of at least two nucleic acid fragments of a double stranded nucleic acid, comprising:
(a) exposing the double stranded nucleic acid to nicking conditions in which, for each strand of the double stranded nucleic acid, at least one linkage between adjacent nucleotides is disrupted at any random location to create a nick while the corresponding nucleotides opposite the nick in the opposing strand of the nucleic acid remain linked wherein the opposing strands remain attached through base pairing interactions, thereby generating a nicked nucleic acid; and
(b) nick translating at least one nick in each strand of the nicked nucleic acid whereby the nick is positioned in sufficiently close proximity to a nick in the opposing strand to generate at least one double-stranded break in the nicked nucleic acid, thereby producing a plurality of nucleic acid fragments of the double stranded nucleic acid, wherein the nicking and/or the nick translating is conducted in the presence of at least one single-strand nucleic acid binding protein, wherein the presence of the at least one single-stranded nucleic acid binding protein improves the yield of fragmented nucleic acids and/or reduces the formation of nucleic acid fragments having rearranged portions; and
(c) ligating the plurality of nucleic acid fragments to adapters.
2 . The method of claim 1 , wherein the nick translating comprises a 5′ to 3′ DNA polymerization/degradation reaction or a 5′ to 3′ DNA polymerization/strand displacement reaction.
3 . The method of claim 1 , wherein the nick translating includes polymerizing one or more unlabeled nucleotides onto the 3′ end of at least one nick.
4 . The method of claim 1 , wherein at least one of the nucleic acid fragments is not labeled.
5 . The method of claim 1 , wherein substantially all of the nucleic acid fragments are not labeled.
6 . The method of claim 1 , further comprising adjusting the average size of the nucleic acid fragments by modulating the reaction conditions for the nicking conditions.
7 . A method for preparing a library of nucleic acid fragments, comprising:
(a) providing a solution including a plurality of double stranded nucleic acid molecules which includes a first and a second double stranded nucleic acid molecule; and
(b) subjecting at least two different nucleic acid molecules of the plurality to the method of claim 1 .
8 . The method of claim 7 , further including fragmenting the at least two different nucleic acid molecules of the plurality in the same reaction mixture.
9 . The method of claim 1 , wherein the nicking conditions include the presence of DNase I.
10 . The method of claim 9 , wherein the nicking conditions further include the presence of magnesium.
11 . The method of claim 9 , wherein the nicking conditions do not include the presence of manganese.
12 . The method of claim 1 , wherein the at least one single-strand nucleic acid binding protein comprises T4 gp32 protein.
13 . The method of claim 1 , wherein the preparation of the library of at least two nucleic acid fragments is catalyzed by one or more enzymes.
14 . The method of claim 1 , wherein the preparation of the library of at least two nucleic acid fragments generates a population of nucleic acid fragments that are selected to have a size range from about 50 bp to about 150 bp, or a size range from about 150 bp to about 250 bp, or a size range from about 250 bp to about 500 bp, or a size range from about 500 bp to about 750 bp, or a size range from about 750 bp to about 1000 bp, or a size range from about 1 kb to about 2 kb, or a size range from about 2 kb to about 5 kb, or a size range from about 5 kb to about 8 kb, or a size range from about 8 kb to about 10 kb, or a size range from about 10 kb to about 20 kb, or a size range from about 20 kb to about 40 kb, or a size range from about 40 kb to about 60 kb, or a size range from about 100 bp to about 250 bp, or a size range from about 100 bp to about 300 bp, or a size range from about 0.8 kb to about 1.4 kb, or a size range from about 200 bp to about 300 bp, or a size range from about 50 bp to about 250 bp, or a size range from about 1 kb to about 5 kb, or a size range from about 5 kb to about 10 kb, or a size range from about 10 kb to about 25 kb, or a size range from about 50 kb to about 60 kb, or a size range from about 100 bp to about 60 kb.