Dear all,

This is related to a ISSR marker based study for generating species specific fingerprinting and developing PCR primers for differentiating two distinct plant species. The aim was to design specific PCR primers from the sequence of species specific fragments generated by ISSR amplification. Differential fragments were purified and cloned in Qiagen pDrive vector (U/A cloning) and were sequenced with vector primers (M13F & M13R). However, while aligning the sequences, it was noted that sequences generated by the forward sequencing primer are present in two different orientation. My query is how to determine the actual orientation of the sequence, so that PCR primers can be developed? Any help, guidance or reference material will be appreciated..

regards,

S D