Ideally a DNA profile will have high Peak Heights (PHs), balanced Peak Height Ratios (PHRs) and no drop out. However, low template DNA (LTDNA) is limited in quantity, quality or both and LTDNA profiles do not always consistently provide interpretable signal. Post-Polymerase Chain Reaction (PCR) purification is an efficient enhancement method that can be utilized to increase the amount of information in a LTDNA profile without elevated stutter, which can be common with other enhancement methods. Post-PCR purification decreases the remaining components left over from amplification, such as deoxyribonucleotide triphosphates, primers, salts, and enzymes so there is less competition during the electrokinetic injection and more DNA will go into the capillary. When post-PCR purification is used; PH’s will increase, PHRs should remain stable before and after purification and may result in recovery of alleles that previously had dropped out. Allele recovery may be the difference between an inconclusive result and an inclusion or exclusion.
The efficacy of Post-PCR purification was assessed by amplifying single source DNA with both ABI AMPFℓSTR® Identifiler® (template mass down to 0.0625 ng) and Identifiler® PLUS (template mass down to 0.03125 ng) and performing post-PCR purification with Qiagen® MinElute® and Macherey-Nagel NucleoSpin®. The original amplified product and purified product were analyzed and compared and it was determined post-PCR purification reduced the primer front, increased the PHs, recovered additional alleles and did not affect the PHRs. On average the Fold Increase (FI) for Identifiler® product purified with Qiagen® MinElute® is 3.5 and the average FI for Identifiler® PLUS product purified with Qiagen® MinElute® and Macherey-Nagel NucleoSpin® is 3.2. Additionally, the stutter percentage observed in the original sample profile was compared to the purified samples to determine if purification affected the stutter percentage of Identifiler® PLUS product. It was determined at only a few alleles the amplified product was above or below the stutter percentage of purified product. The stutter percentage values for purified samples were further compared to the Identifiler® PLUS manual and only one allele’s stutter percentage is above the companies stutter cut off values. Post-PCR purification with Qiagen® MinElute® or Macherey-Nagel NucleoSpin® is a successful enhancement method to increase information of a LTDNA profile without introducing additional complications that other enhancement methods are known to do.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/21191 |
| Date | January 2013 |
| Creators | Kinnaman, Emily Allison |
| Publisher | Boston University |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | This work is being made available in OpenBU by permission of its author, and is available for research purposes only. All rights are reserved to the author. |
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