An undergraduate laboratory experiment for upper-level forensic science, biochemistry, or molecular biology courses: Human DNA amplification using STR single locus primers by real-time PCR with SYBR green detection

摘要

In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The primer DNA concentration was evaluated using UV-vis spectroscopy and dilutions were prepared for PCR based upon the absorbance at 260 nm and the theoretical extinction coefficient. In a previous lab, students were instructed in the use of the NCBI Web site to locate genes of interest, PubMed to search for primers, and Web tools to design primers; they were instructed in PCR theory in the paired lecture portion of the course and in previous laboratory sessions. As there are no educational kits for demonstrating real-time PCR, recently published TPOX primers directed at the forensic field in lieu of more-costly DNA quantification kits and purchased student-designed primers were used in conjunction with the iQ SYBR Green Supermix from Bio-Rad. Students pipetted the K562 standard DNA template, the primers, and the reaction mix into a 96-well plate. With the help of the instructor, they programmed the Bio-Rad iQ5 instrument to do gradient PCR. The instructor recovered the plate and data at the completion of the 3-h PCR experiment. In a subsequent laboratory session, the students ran agarose gels of the PCR products to confirm the length of the amplicons with a DNA ladder and analyzed the real-time PCR results. This laboratory was taught to the students enrolled in the second of a sequence of upper-level criminalistics undergraduate classes. Students interested in pursuing post-graduate study and research, technical work in an academic biochemistry or molecular biology or clinical laboratory, or employment as a DNA analyst in the crime lab setting will benefit most by this opportunity. This lab would also be suitable for upper-level biochemistry and molecular biology laboratories.