Creation of DNA overhangs by using modified DNA overhang cloning method.
DNA overhang cloning (DOC) method was invented by Kevin A. Jarrell and colleagues to create DNA overhangs at the ends of polymerase chain reaction (PCR) fragments. To increase the ligation products of DOC and make it possible to join the ligation products together to yield a larger DNA fragment without amplification by using the secondary PCR, we modified the original method by lengthening RNA-DNA hybrids to 8 bp, using RNase H to digest RNA chains, and so on. We found that the ligation of PCR fragments with DNA overhangs could be improved significantly by using the modified DOC. The ligation products of the modified DOC were rich enough to be separated by using agarose gel and joined together to yield a larger DNA fragment without amplification by using the secondary PCR. The modified DOC is simpler and much more effective than the original one. The creation of DNA overhangs by using complementary DNA oligonucleotides, T4 ligase, and RNase H is simpler and cheaper than that by using reverse transcriptase and RNase H.
Li
Wei
W
Laboratory of Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, People's Republic of China. liwei60@yahoo.com
Gao
Feng
F
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; D011088:DNA Ligases