Database of primers of genes in Escherichia coli K-12 without recognition sequence of NdeI and BamHI

Polymerase chain reaction (PCR) remains a critical tool for molecular biologists whether in amplifying small quantities of DNA from the environment or single cell, or in cloning of genes from one species into another. The process requires short stretches of nucleotides known as primers to help identify the region of the template DNA for polymerase amplification. Primers designed for amplification of target genes from environmental DNA differs from those designed for molecular cloning. In the case of molecular cloning, key characteristics of the primer include (i) the need for both forward and reverse primers for targeting a specific stretch of template DNA for amplification, (ii) the primers must also be able to form complementary base pairing with a short stretch of template DNA (18 to 25 nucleotide) with melting temperature of about 55 ^oC, (iii) recognition sequence for specific restriction enzymes selected for molecular cloning should also be appended to the start of the primer sequence, and (iv) a flanking sequence is also needed to be appended upstream of the restriction enzyme recognition sequence to help hinge the polymerase to the DNA-primer construct. Using an in-house MATLAB software, a primer database was created for all genes in Escherichia coli K-12 strain MG1655 (Genbank accession number, NC_000913.3) not having recognition sequence of restriction enzymes, NdeI and BamHI, which otherwise would result in the digestion of the gene by the restriction enzyme used and the failure of the cloning effort. Primers designed by the MATLAB algorithm fulfilled the requirements described above for primers to be used in molecular cloning. For each gene in the genome of E. coli K-12 MG1655 without recognition sequence of NdeI and BamHI, one forward and reverse primer was designed for cloning the gene using the restriction enzyme set, NdeI and BamHI. Collectively, a primer database was created for all genes in E. coli K-12 strain MG1655 not having recognition sequence of NdeI and BamHI. Primers designed could be used for cloning the respective genes using NdeI and BamHI as recognition sequences for the two restriction enzymes have been appended to the primer sequence. Overall, the primer database should find use as a reference for researchers in molecular cloning and plasmid construction applications involving E. coli K-12 MG1655.