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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Use of a direct lysis procedure to assess DNA recovery using manual and robotic DNA extraction methods

Uramoto, Kyra Miyuki 09 June 2023 (has links)
DNA (Deoxyribonucleic Acid) must be extracted from the cell before it can be amplified using the PCR (Polymerase Chain Reaction) to generate an STR (Short Tandem Repeat) profile. Although extraction methods such as binding DNA to silica remove PCR inhibitors, they have complex procedures that can lead to partial DNA loss. Evaluating the efficiency of an extraction method can be difficult, as the original amount of input DNA may be unknown. Knowledge of the amount of DNA expected to be present in a sample can provide quantitative information on an extraction method’s efficiency, as the recovery of DNA from a DNA extraction process can be calculated using the expected DNA value. Theoretically, while using a direct lysis extraction method, all the cells in the sample are lysed and present in that single tube, so quantitative PCR (qPCR) values of samples extracted using a direct lysis method such as forensicGEMTM can be used as an expected yield value. Validation studies determine if a method is reliable, reproducible, and robust. An internal validation study, following the Federal Bureau of Investigation’s Quality Assurance Standards (QAS) for Forensic DNA Analysis Methods, was performed for the Maxwell® FSC instrument, a robotic DNA extraction instrument designed to remove PCR inhibitors from forensic samples. Results from this study were compared to results obtained on a previously validated Maxwell® 16 instrument. The validation study was successful, as the study demonstrated the instrument could process known/non-probative evidentiary samples, is sensitive, precise, accurate, can process mixture samples, and had no detectable contamination in the process. An added study, done post validation, compared the amount of DNA obtained using a “direct lysis method” with the amount of DNA obtained using samples equivalent to those used in the instrument validation. However, upon comparing the amount of DNA recovered using the Maxwell® FSC instrument to the amount of DNA obtained using a direct lysis method, which in theory would recover 100% of the DNA, the percent yield for serially diluted blood samples was low. Only 10.79% of the DNA was recovered for the 1:10 dilution, 14.44% was recovered for the 1:100 dilution, and 8.00% was recovered for the 1:1000 dilution. The DNA IQTM System – Small Sample Casework kit uses the same chemistry and the same reagents as the Maxwell® FSC DNA IQTM Casework Kit. To provide additional data on DNA recovery, the Maxwell® FSC Study was replicated using the DNA IQTM System – Small Sample Casework Kit, following the manufacturer’s instructions for manual extractions. The study showed that about 77.71% of the DNA is lost when using neat samples, 96.88% is lost for the 1:10 dilution samples, 95.22% is lost for 1:100, and 99.00% for 1:1000. Steps from the DNA IQTM System – Small Sample Casework Protocol were identified where, potentially, the DNA loss could have occurred. These included DNA left in the waste products, DNA left on the swatch, and DNA left on the Resin after elution. Study data identify experimentally two major sources of DNA loss: the waste and the swatch. When quantifying the “waste,” for almost every individual sample, more DNA remained in the waste than was found in the initial extract using the DNA IQTM System – Small Sample Casework Kit. The high amounts of DNA in the waste indicates that the DNA IQ™ Resin does not bind to all the available nucleic acid in the solution. The data and other data characterizing DNA loss at particular steps of the protocol are described in this study. Modifications to the protocol are described, which were made, in an attempt, to increase the amount of DNA bound to the Resin that could subsequently be recovered. Laboratories that use DNA IQTM reagents need to be aware that a significant amount of the sample may be lost when following the procedure. Preliminary results indicate that yield can be improved by modifying the procedure to either increase the Resin volume, increase the incubation temperature, or recapture DNA from the first waste.

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