Ligation enhancement

Patent metadata

Field	Value
Publication number	US-9499811-B2
Application number	US-201414190747-A
Country	US
Kind code	B2
Filing date	Feb 26, 2014
Priority date	May 6, 2011
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.

First claim

What is claimed is:

1. A method of enhancing ligation between nucleic acid fragments, the method comprising:
(a) mixing a composition comprising T4 DNA ligase and a ligase reaction buffer comprising one or more small molecule diol ligation enhancers having a molecular weight of less than 500 daltons at a concentration of 1%-50% v/v, with a plurality of nucleic acid fragments in a ligation buffer; and
(b) permitting ligation wherein the efficiency of ligation is enhanced by at least 25% compared to the efficiency of a ligation in the absence of the small molecule enhancer.

2. A method according to claim 1 , wherein ligation is intramolecular.

3. A method according to claim 1 , wherein ligation is intermolecular.

4. A method according to claim 1 , wherein the plurality of nucleic acid fragments are double-stranded DNA with blunt ends, single-base overhangs or staggered ends.

5. A method according to claim 1 , wherein the efficiency of ligation is enhanced by at least 4-fold as determined by electroporation of host cell.

6. The method of claim 1 , wherein the one or more small molecule diol ligation enhancers are selected from the group consisting of 1,2-propanediol (1,2-PrD), 1,3-propanediol (1,3-PrD), 2-methyl-1,3-propanediol, 2,2-dimethyl-1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,9-nonanediol, 1,12-dodecanediol, 1,3-butylethylpropanediol, methylpropanediol, and methylpentanediols.

Assignees

• New England Biolabs Inc

Inventors

• Kucera Rebecca
• Evans Jr Thomas C

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title