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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23185 43419 2014 6 PDF Available
Title
A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences
Abstract

•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 4–5 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.

Keywords
WGA, whole genome amplification; LM-PCR, ligation-mediated polymerase chain reaction; MDA, multiple displacement amplification; mLM-PCR, modified LM-PCR; PEP, primer extension pre-amplification; DOP-PCR, degenerate oligonucleotide primed PCR; RAs, represe
First Page Preview
A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 184, 20 August 2014, Pages 1–6
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering