Simple innovative adaptor to improve genome walking with convenient PCR

Background Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor usingMAP30gene sequence ofMomordica charantiaplant as a model to improve genome walking with convenient PCR. Results The adaptor was designed using multiple restriction sites ofHindIII,BamH I,EcoR I, andBglII enzymes with no restriction site in a known sequence of theMAP30gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5 ' phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5 ' phosphate group. Conclusion PCR was done to amplify the unknown sequence usingMAP30gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.