Open AccessImprovement of multiplex semi-nested PCR system for screening of rare mutations by high-throughput sequencing
Author(s) -
Yingchun Zhang,
Xu Chi,
Laibao Feng,
Xinqi Wu,
Xiaoquan Qi
Publication year - 2019
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2019-0001
Subject(s) - multiplex , biology , genetics , mutation , computational biology , mutant , multiplex polymerase chain reaction , crispr , nested polymerase chain reaction , population , cas9 , dna sequencing , gene , polymerase chain reaction , demography , sociology
The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced rice population. However, this strategy has the potential for further optimization. Here, we describe an improved multiplex semi-nested PCR target-enrichment strategy with simplified processing procedures, reduced false-positive rates and increased mutation detection frequency (1 mutation/73 Kb).