Abstract：
:In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), split marker deletion method, co-transformation of single-stranded DNA and mutant Cre-loxP system. Using a slightly modified LiAc/SS-DNA/PEG procedure, the co-transformation double-stranded split marker constructs together with single-stranded split marker constructs resulted in at least 70% homologous recombination events when the homologous genomic DNA fragment had a size of approximately 500bp. Our evidence suggested that single-stranded DNA may be responsible for the increased gene disruption efficiency. We demonstrated the effectiveness of the method for gene disruption by constructing both single and double gene disruptions at the ALG3 and URA5 loci in the same genetic background. The method described here presents an improved strategy for gene disruption and a potential application for investigation of biological processes in other yeast strains.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Qian W,Song H,Liu Y,Zhang C,Niu Z,Wang H,Qiu Bdoi
10.1016/j.mimet.2009.09.004subject
Has Abstractpub_date
2009-12-01 00:00:00pages
253-9issue
3eissn
0167-7012issn
1872-8359pii
S0167-7012(09)00274-7journal_volume
79pub_type
杂志文章abstract：:Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indi...
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