I am stumped, and would appreciate any help on this. Here is the situation:
I am working on PhyA from S.ruminantium, and am interested in a construct containing the Asp223 to Asn mutation. The sequence encoding the mature region of PhyA from S. ruminantium (nt 312 to 1268; Genbank accession number AF177214) was amplified using primers containing the recognition sequence for BamHI. The PCR product was digested using BamHI, and DNA ligated into similarly cleaved pQE30 (Qiagen). The presence of the desired expression construct was confirmed by restriction digestion and nucleotide sequence analysis.
My questions are this:
1. How can I design primers to create an expression construct containing the D223N mutation?
2. How can I introduce a unique restriction site into the PCR product that can be used in restriction profiling to confirm amino acid change? I want to be able to see the differences in profiles on agarose, so they need to be large.