Streptavidin-biotin binding of DNA amplicons: methods for the typing and re-typing of forensically relevant short tandem repeats
Shea, Jessica Anna-Marie
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Submission of evidentiary samples to DNA units for exhaustive testing is becoming commonplace. For these samples, only one attempt at amplification is possible. However, more than one amplification may be necessary if the condition of the DNA causes poor amplification, more than one type of STR kit testing is required, or if there is an instrument malfunction during amplification. Current research into the re-amplification of already amplified samples focuses on placing the PCR product back into the thermal cycler with new reagents for additional cycles. These methods typically result in outcomes which are unsatisfactory for forensic purposes. As a result, there is a need for a forensic method capable of recovering the original template DNA for purposes of re-amplification. This study outlines the development of a novel method to recover the original template DNA in a condition that allows for re-amplification using new STR loci. A dynamic model was designed to assist in the experimental optimization. Amplification was performed using biotinylated primers and the post PCR 'work product' was subsequently cleaned using streptavidin coated magnetic beads to remove the STR amplicons. Centrifugal filtration followed in order to remove any remaining primers and salts that may interfere with re-amplification. Re-amplification was then performed with non-biotinylated primers. Re-amplification of the template DNA using a new STR locus was successful, making the amplification of limited DNA samples non-destructive and the notion of 'exhaustive DNA typing' obsolete.
Thesis (M.S.)--Boston University