A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences

•Obtained unbiased WGA with high efficiency by using type IIS restriction enzymes.•Self-ligation problem was solved by producing more than 256 kinds of sticky ends.•Realized WGA by using adapters with random overhangs and single universal primer.•Amplification efficiency was evaluated by quantitativ...

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Bibliographic Details
Published in:Journal of biotechnology 2014-08, Vol.184, p.1-6
Main Authors: Pan, Xiaoming, Wan, Baihui, Li, Chunchuan, Liu, Yu, Wang, Jing, Mou, Haijin, Liang, Xingguo
Format: Article
Language:English
Subjects:
Amplification
Constrictions
Deoxyribonucleic acid
DNA Ligases - genetics
DNA Restriction Enzymes - genetics
Enzymes
Fragments
Gene detection
Gene sequencing
Genome, Bacterial
Genomes
Ligation-mediated PCR
Nucleic Acid Amplification Techniques
Overhang
Polymerase Chain Reaction - methods
Self-ligation
Vibrio parahaemolyticus
Whole genome amplification
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Summary:•Obtained unbiased WGA with high efficiency by using type IIS restriction enzymes.•Self-ligation problem was solved by producing more than 256 kinds of sticky ends.•Realized WGA by using adapters with random overhangs and single universal primer.•Amplification efficiency was evaluated by quantitative PCR. 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.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2014.04.020