Journal
BMC BIOINFORMATICS
Volume 14, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/1471-2105-14-105
Keywords
Site-directed mutagenesis; SDM; PCR; Cloning; Restriction endonucleases; Oligonucleotides; Software
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Funding
- Microsoft Research through the PhD Scholarship Programme
- BBSRC grant [BB/HO24867/1]
- BBSRC [BB/H009817/1, BB/F001630/1, BB/K015893/1, BB/H024867/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/K015893/1, BB/F001630/1, BB/H009817/1, BB/H024867/1] Funding Source: researchfish
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Background: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results: We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest. Conclusions: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.
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