A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences.
Ligation-mediated polymerase chain reaction (LM-PCR) is a whole genome amplification (WGA) method, for which genomic DNA is cleaved into numerous fragments and then all of the fragments are amplified by PCR after attaching a universal end sequence. However, the self-ligation of these fragments could happen and may cause biased amplification and restriction of its application. To decrease the self-ligation probability, here we use type IIS restriction enzymes to digest genomic DNA into fragments with 4-5nt long overhangs with random sequences. After ligation to an adapter with random end sequences to above fragments, PCR is carried out and almost all present DNA sequences are amplified. In this study, whole genome of Vibrio parahaemolyticus was amplified and the amplification efficiency was evaluated by quantitative PCR. The results suggested that our approach could provide sufficient genomic DNA with good quality to meet requirements of various genetic analyses.
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Xiaoming
X
Pan
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Baihui
B
Wan
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Chunchuan
C
Li
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Yu
Y
Liu
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Jing
J
Wang
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
Haijin
H
Mou
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China. Electronic address: liangxg@ouc.edu.cn.
Xingguo
X
Liang
D011088:DNA Ligases / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics*; D016680:Genome, Bacterial; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D014736:Vibrio parahaemolyticus
D004262:DNA Restriction Enzymes; D011088:DNA Ligases
Gene detection; Ligation-mediated PCR; Self-ligation; Whole genome amplification