Rights statement: © 2013 Karnik et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - SDM-assist software to design site-directed mutagenesis primers introducing "silent" restriction sites
AU - Karnik, Abhijit
AU - Karnik, Rucha
AU - Grefen, Christopher
N1 - © 2013 Karnik et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
PY - 2013/3
Y1 - 2013/3
N2 - 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.
AB - 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.
KW - Site-directed mutagenesis
KW - SDM
KW - PCR
KW - Cloning
KW - Restriction endonucleases
KW - Oligonucleotides
KW - Software
U2 - 10.1186/1471-2105-14-105
DO - 10.1186/1471-2105-14-105
M3 - Journal article
VL - 14
JO - BMC Bioinformatics
JF - BMC Bioinformatics
SN - 1471-2105
M1 - 105
ER -