Development and application of a PCR multiplex to assess the quality and quantity of forensic DNA extracts

Iyavoo, Sasitaran (2014) Development and application of a PCR multiplex to assess the quality and quantity of forensic DNA extracts. Doctoral thesis, University of Central Lancashire.

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Isolation of DNA from skeletonised human remains can be problematic. In addition to DNA degradation, enhanced by high temperature and humidity, there are often potent polymerase chain reaction (PCR) inhibitors present within the samples. It is therefore important to extract the maximum amount of available DNA whilst removing any amplification inhibitors that may be present.

Whilst real-time PCR methods are available for quantification and detection of PCR inhibitors the information received is limited as real-time PCR targets amplicons that are much smaller than those typically targeted in forensic analysis. To gain more information on the quality of extracted DNA a new multiplex PCR assay comprising a 4-plex targeting amplicons of 70 base pairs (bp), 194 bp, 305 bp and 384 bp along with two Internal Amplification Contols (IACs) of 90 bp and 410 bp was developed. This multiplex was optimised so that it worked with template amounts ranging between 0.10 ng and 200 ng; partial profiles were obtained with as little as 0.02 ng. The IACs were effective in detecting PCR inhibitors.

The multiplex also assessed as a quantification tool. Plotting peak height compared to input DNA of a standard dilution series produced a coefficient of determination (R2) of 0.8308. The multiplex was found to provided reasonable estimates of DNA concentration, when the sample concentration was between 12.5 – 100 ng; relative standard deviations were all below 10% in this range for 30% of tested samples. However, real-time PCR proved to be more precise and was used in the rest of the study for the purposes of quantification.

In forensic cases bones and teeth often provide some of the most challenging samples to extract good quality DNA. Using the optimised multiplex to assess the quality of DNA extracts five extraction methods: ChargeSwitch® gDNA Plant Kit, DNA IQTM System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol extaction methods were assessed for their capability for extracting clean DNA from bone samples. Prior to the main experimentation several evaluation studies were carried out to optimise the methods being used. Based on the results, decalcification was not used for any of the extractions as non-decalcified extracts contained higher amounts of DNA. For the phenol-chloroform-isoamyl alcohol extraction it was determined that whilst ethanol precipitation provided higher amounts of DNA, the extracts using Amicon 30kDa filters (Amicon ultra-0.5 centrifugal filter unit with ultracel-30 membrane) were cleaner. Based on poor results with degraded bone samples a pre-process technique was developed; these extractions started with 250 mg of pulverised bone sample which was then concentrated and cleaned up using Amicon 30kDa filters (Amicon ultra-2 ml centrifugal filters for DNA purification and concentration) before carrying out the standard extraction procedures.

After optimisation of the extraction methods the comparison study showed that the phenol-chloroform-isoamyl alcohol extraction method produced the highest DNA yields with both fresh and degraded bone samples, followed by DNeasy® Blood & Tissue Kit, ChargeSwitch® gDNA Plant Kit, PrepFiler® BTA Forensic DNA Extraction Kit and DNA IQTM System Kit. However, all produced DNA that could be amplified and did not contain any inhibition.

Another application of the multiplex was to assess the effectiveness of different DNA preservation methods by examining the amount and quality of DNA recovered after preservation. Five methods: cell lysis solution (with 1% sodium azide), dehydration / freeze drying, ethanol (96%), freezing and room temperature storage were used to study the effectiveness of preservation methods on fresh and three-month old decomposed pig bone samples which were preserved for 6 weeks, 6 months and 1 year. The results showed that freezing is the best preservation method for both fresh and degraded bone samples for long-term storage followed by ethanol (96%), dehydration / freeze drying and room temperature storage. However, full profiles were obtained from both fresh and degraded bone samples from all methods, except cell lysis solution (with 1% sodium azide). Cell lysis solution (with 1% sodium azide) preservation method tended to be good for short-term storage but with the long-term preservation, less DNA yield was obtained and also the electropherograms showed higher levels of DNA degradation.

Finally, using the optimised DNA extraction methods, the multiplex was tested using forensic samples comprising of 30 bone samples from casework in Malaysia and simulated body fluid evidences subjected to environmental insult in the United Arab Emirates. The application illustrated the effectiveness of the multiplex to identify PCR inhibitors and identify DNA degradation, providing supplementary information to real-time PCR.

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