Clone Sequence ValidationOverviewA. Use of SURVEYOR Nuclease for the detection of PCR errors or mutations in plasmids cloned in bacteria Identification of error-free clones
The use of PCR techniques for the generation and subsequent cloning of genomic or cDNA inserts into plasmid vectors is widely accepted. However, clones obtained with these methods may contain sequence errors introduced during the PCR amplification process itself. The identification of clones that might harbor plasmids with sequence errors is achieved by employing SURVEYOR Nuclease, a mismatch-specific endonuclease derived from celery. PCR products derived from plasmid DNA in bacterial colonies are combined and hybridized with PCR products derived from primary cDNA or genomic DNA sequences. Any sequence differences between the two types of PCR products will give rise to mismatches that are recognized and cleaved by SURVEYOR Nuclease and subsequently analyzed by agarose gel electrophoresis. An outline of the process is shown below. In this context, the Control Amplicons derived from the Wild-type DNA could be represented by PCR products derived from source of cDNA obtained by reverse transcriptase mediated cDNA synthesis, from genomic DNA or from plasmid DNA in a reference clone. |