Ligation enhancement
View Patent ↗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.
1. 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, the ligase reaction buffer being capable of enhancing, by at least 25%, intramolecular or intermolecular ligation of a polynucleotide fragment or fragments.
2. A composition according to claim 1 , wherein the one or more small molecule diol ligation enhancers are selected from optionally substituted straight or branched chain diols containing 2 to 20 carbons.
3. A composition according to claim 1 , wherein the one or more small molecule diol ligation enhancers are selected from the group consisting of: 1,2-PrD, 1,3-PrD, 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol.
4. A composition according to claim 1 , wherein the small molecule diol enhancer is 1,2-PrD.
5. A composition according to claim 1 , further comprising at least two nucleic acid fragments wherein the nucleic acid fragments are double-stranded DNA having blunt ends for ligation to each other.
6. A composition according to claim 1 , further comprising at least two nucleic acid fragments where the nucleic acid fragments are double-stranded DNA having single-base overhangs for ligation to each other.
7. A composition, according to claim 1 , comprising: 0.01-200 units/0 restriction endonuclease, 0.01-2000 units/0 ligase, 0.01-200 units/0 polynucleotide kinase, in a buffer optionally containing PEG 5000-10,000.
8. A composition according to claim 7 , wherein the one or more small molecule diol ligation is 1,2-PrD and the composition further comprises glycerol.
9. A composition comprising T4 DNA ligase and one or more small molecule diol ligation enhancers having a molecular weight of less than 500 daltons, wherein the one or more small molecule diol ligation enhancers are selected from optionally substituted straight or branched chain diols containing 2 to 20 carbons and the optionally substituted straight or branched diol is 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.
10. A composition comprising T4 DNA ligase and one or more small molecule diol ligation enhancers having a molecular weight of less than 500 daltons, wherein the amount of the small molecule diol enhancer is in the range of 1%-20% v/v.
11. A composition according to any of claim 1 , 2 - 9 , 3 , 4 , 10 - 6 or 7 - 8 wherein the composition does not contain polyethylene glycol (PEG).