Validation of a DNA extraction method using diluted ATL as an extraction buffer with the Qiagen® Lyse&Spin Basket Kit
Cole, Kelsey Ann
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The ability to confidently obtain deoxyribonucleic acid (DNA) profiles from a variety of sample types in a crime laboratory is very important. The first step in the analysis of DNA in a forensic crime laboratory is the extraction of the nucleic acid material from the rest of the cellular material contained in the sample. There are many different extraction methods available for use in the field of forensics but one that is reliable, cost effective, and easy to use is necessary. One of the methods that meet these requirements is a solid-phase extraction utilizing a silica membrane that binds the DNA in the presence of chaotropic salts. This solid-phase extraction using a silica membrane is ideal for automation and use with a bio-robot. One commonly used instrument is the BioRobot EZ1® system (Hilden, Germany) from Qiagen®. Automation using these robots was the first step in decreasing the time it takes to perform extraction as well as reduce the potential for contamination, but there are still opportunities to improve in both of these areas. In this study the Qiagen® Lyse&Spin Basket Kit was evaluated due to its potential to further decrease the time needed for extraction and the eliminate a step of the extraction process where contamination could be introduced. Laboratories around the country have reported problems with using Buffer G2 as an extraction buffer when used with samples such as blood on fabric. The reason for this is currently unknown, but in order to continue to confidently extract samples of this type, a change to diluted ATL buffer was suggested. The current extraction buffer, G2, used in the extraction protocol at the Kansas City Police Crime Laboratory was not yielding results in some situations such as blood on fabric. Switching to diluted ATL yielded the same quantity of DNA and the same quality of DNA profiles with mock casework samples. When diluted ATL was used with Quality Control (QC stain) samples it yielded significantly more DNA during extraction. When the Qiagen® Lyse&Spin Basket Kit was used during the extraction of whole blood on fabric (QC stain samples) they showed a significantly lower quantitation value than the tubes currently used in the Kansas City Police Department Crime Laboratory. The same results were obtained when the Qiagen® Lyse&Spin Basket Kit was used to extract 1:2 diluted saliva samples. When samples with lower quantities of DNA, such as 1:100 blood dilutions, 1:50 saliva dilutions, and mock casework samples, were examined the Qiagen® Lyse&Spin Basket Kit there were no significant differences seen when compared to the currently used baskets. When the quantitation data was analyzed for the QC stain samples extracted with the Qiagen® Lyse&Spin Basket Kit, abnormally high degradation index (DI) values were observed. It was determined that these high values did not affect the integrity of the sample. In order to determine the possible cause behind the poor performance of the Qiagen® Lyse&Spin Basket Kit when used to extract samples with higher starting amounts of DNA an experiment was designed to determine if genetic material was left on the cotton swatch in the spin basket. It was seen that DNA was left behind on the cotton swatches from both the Qiagen® Lyse&Spin Baskets and the currently used baskets. It appeared that more DNA was contained on the fabric used with the Qiagen® Lyse&Spin Basket, but further research is needed to determine if this difference is significant.