CHEMICALLY MODIFIED LIGASE COFACTORS, DONORS AND ACCEPTORS
Provided herein are methods for ligation of polynucleotides containing modified ligation components, particularly modified ligase cofactors, modified acceptors and modified donors. The methods readily applied to ligation-based assays for detection of a nucleic acid sequence where the use of the modified cofactor improves discrimination between matched and mismatched templates. Furthermore, the use of the modified ligation components reduces or eliminates the ligation in the absence of nucleic acid template. In addition, methods are applied to the preparation of nucleic acid libraries using modified acceptor probes and modified donor probes that reduce or eliminate probe dimerization during the ligation process.
1 . A method for preparing a RNA nucleic acid library that reduces or inhibits acceptor:donor probe dimer formation, said method comprising:
incubating RNA target nucleic acid fragments in a reaction mixture comprising one or more RNA ligase, a modified donor probe, and a modified acceptor probe to prepare a RNA nucleic acid library,
wherein said RNA nucleic acid library contains RNA target nucleic acid fragments comprising said modified donor probe ligated to the 3′-terminus and said acceptor probe ligated to the 5′terminus, and
wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited.
2 . A method for preparing a RNA nucleic acid library that reduces or inhibits acceptor:donor probe dimer formation, said method comprising:
incubating RNA target nucleic acid fragments in a reaction mixture comprising an RNA ligase and an adenylated modified donor probe in the absence of ATP to produce a 3′-modified RNA target nucleic acid; and
incubating said 3′-modified RNA target nucleic acids with a modified acceptor probe and RNA ligase to produce said RNA nucleic acid library,
wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited.
3 . The method according to claim 1 , wherein said RNA target nucleic acid fragments are incubated with a 5′-phosphorylated modified donor probe, a modified acceptor probe and an RNA ligase in the presence of ATP.
4 . The method according to claim 1 , wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited by about 5% to about 100% relative to dimerization of an unmodified donor probe to said unmodified acceptor probe.
5 . The method according to claim 1 , wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited by about 75% to about 100% relative to dimerization of an unmodified donor probe to said unmodified acceptor probe.
6 . The method according to claim 1 , wherein said modified donor probe and said modified acceptor probe are ligated onto said RNA target nucleic acids with similar efficiency to ligation onto said RNA target nucleic acids with an unmodified donor probe and an unmodified acceptor probe.
7 . The method according to claim 6 , wherein said efficiency is about 5% to about 200%.
8 . The method according to claim 6 , wherein said efficiency is about 50% to about 200%.
9 . The method according to claim 6 , wherein said efficiency is about 50% to about 150%.
10 . A method for preparing a DNA nucleic acid library that reduces or inhibits acceptor:donor probe dimer formation, said method comprising:
incubating DNA target nucleic acid fragments in a reaction mixture comprising a DNA ligase and a double stranded modified donor probe and a double stranded modified acceptor probe to prepare a DNA nucleic acid library,
wherein dimerization of said double stranded modified donor probe to said double stranded modified acceptor probe is reduced or inhibited.
11 . The method according to claim 10 , wherein said double stranded modified donor probe and double stranded modified acceptor probe comprise a 5′-phosphate group or a 5′-adenylate group.
12 . The method according to claim 11 , wherein said double stranded modified donor stand comprises said 5′-phosphate group and said 5′-adenylate group.
13 . The method according to claim 10 , wherein said double stranded modified donor probe and double stranded modified acceptor probe have blunt ended termini.
14 . The method according to claim 10 , wherein said double stranded modified donor probe and double stranded modified acceptor probe have T-tailed termini.
15 . The method according to claim 10 , wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited by about 5% to about 100% relative to dimerization of an unmodified donor probe to an unmodified acceptor probe.
16 . The method according to claim 10 , wherein dimerization of said modified donor probe to said modified acceptor probe is reduced or inhibited by about 75% to about 100% relative to dimerization of an unmodified donor probe to an unmodified acceptor probe.
17 . The method according to claim 10 wherein the modified donor probe and modified acceptor probe are ligated onto said DNA target nucleic acid fragments with similar efficiency to ligation onto DNA target nucleic acid fragments with an unmodified donor probe and an unmodified acceptor probe.
18 . The method according to claim 17 , wherein said efficiency is about 55 to about 200%.
19 . The method according to claim 17 , wherein said efficiency is about 50-200%.
20 . The method according to claim 17 , wherein said efficiency is about 50-150%.
21 . A kit for preparing a nucleic acid library, said kit comprising instructions for performing said library preparation, a modified donor probe, a modified acceptor probe, a cofactor dependent ligase and a ligase cofactor.