TY - JOUR
T1 - Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening
AU - Huang, Chunyi George
AU - Agre, Peter
AU - Strange, Kevin
AU - Lamitina, Todd
N1 - Funding Information:
This work was supported by National Institutes of Health (NIH) grants DK51610 and DK61168 and a pilot project grant supported by the Vanderbilt O’Brien Center (DK39261) to K.S., by NIH grants HL33991, HL48268, and EY11239 to P.A., and by a research fellowship from the National Kidney Foundation to T.L. The authors declare no competing interests in this work.
PY - 2006/1
Y1 - 2006/1
N2 - The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.
AB - The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.
KW - Ethylnitrosourea
KW - Mutation
KW - Polymerase chain reaction
UR - http://www.scopus.com/inward/record.url?scp=29644431851&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=29644431851&partnerID=8YFLogxK
U2 - 10.1186/1471-2105-7-83
DO - 10.1186/1471-2105-7-83
M3 - Article
C2 - 16382185
AN - SCOPUS:29644431851
SN - 1073-6085
VL - 32
SP - 83
EP - 86
JO - Molecular Biotechnology
JF - Molecular Biotechnology
IS - 1
ER -