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

作者：Pan Xiaoming; Wan Baihui; Li Chunchuan; Liu Yu; Wang Jing; Mou Haijin; Liang Xingguo^*

来源：Journal of Biotechnology, 2014, 184: 1-6.

DOI：10.1016/j.jbiotec.2014.04.020

• Whole genome amplification
• Ligation-mediated PCR
• Self-ligation
• Gene detection

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 4-5 nt 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.

• 出版日期2014-8-20
• 单位中国海洋大学