Abstract
The focus of this summer?s project was to synthesize the DNA for a glycosylated version of the human coagulation Factor VIII protein using the same methods used to obtain the DNA for a non-glycosylated version, which includes application of a set of sixteen previously-designed overlapping oligonucleotide primers. We used a recursive PCR technique that allows these DNA fragments to come together, forming a larger DNA fragment with shorter B-domain. By making the B-domain smaller, we believe it will be easier to isolate and characterize the 3-D structure of the Factor VIII protein. Once solving its structure, researchers can create drugs that are more effective than simply injecting human F8 proteins intravenously (the current technique for treating hemophilia). After tweaking concentrations of DNA fragments for the PCR, we have successfully generated a DNA fragment that was shown, using DNA sequencing, to be the desired sequence. Future experiments will involve restriction enzyme digests and ligations into the human Factor VIII vector to create plasmids for protein expression through the use of bacteria and viruses. Once expressed, we can begin spectroscopic characterization the Factor VIII protein itself.
