The development of PCR internal controls (PICs) for forensic DNA analysis

Zahra, Nathalie (2009) The development of PCR internal controls (PICs) for forensic DNA analysis. Doctoral thesis, University of Central Lancashire.

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Proper interpretation of DNA profiles depends on the quality of the DNA samples, the amplification efficiency and the success of post-PCR processing. Chemicals associated with forensic samples can affect the amplification process while random errors occurring during pipetting and electrokinetic injection can cause variability. This can lead to either a
reduced signal or lack of DNA profiles.
As recommended by the SWGDAM, laboratories carrying out DNA profiling have to adopt standardise and validate procedures, which lead to high levels of quality assurance and control. During amplification, monitoring is restricted to the use of exogenous controls, which are unable to identify issues associated with individual samples. To address this
limitation, four PCR Internal Controls (PJC5) i.e. two Internal Amplification Controls (IACs) and two Internal Non-Amplifiable Control (INAC5), to be used with the AmpFtSTR® SGM Plus® kit were developed.
The IACs (90 bp and 410 bp) and INACs (80 bp and 380 bp) fragments were generated from the plasmid pBR322 and added along with human DNA in a 12.5 gl PCR volume.
During the reaction the IAC% and 1AC 41 0 are amplified non-competitively with ROX labelled primers, while the pre-labelled INAC80 and 1NAC 380 were not involved with the amplification process. Both sets of fragments were detected as red peaks on the electropherogram flanking the human DNA profile. To study the behaviour of the markers within the system and their effect on the performance and sensitivity of the assay, the PICs were used during the amplification of human DNA of different quantity and quality and with the addition of three common inhibitors.
Initial experiments involving the individual development of the fragments showed that both the INACs and IACs can be successfully applied to the amplification of human DNA with SGM Plus® reaction under optimised conditions, without significantly impacting the quality of human DNA profile.
As the INACs fragments are designed not to amplify during the reaction, they gave a stable signal that can be used to monitor the post-PCR sample processing. The peak height ratios (PHRs) of human DNA with that of the LNAC8 0 or 1NAC380 can also be used to normalise the signal and assess the amplification efficiency of human DNA samples within replicates of the same sample run under the same conditions.
The JACs on the other hand gave more information on the process of the PCR. They were able to monitor changes in the amplification efficiency and detect presence of inhibitors with a minimum inhibitory concentration closer to the SGM Plus® as compared to the Quantifiler® WC system. The IAC90 and 1AC410 ratios were also used to distinguish between partial profiles obtained from degraded DNA and those resulting from partial inhibition.
Combining the two sets of fragments together with the amplification of human DNA needed further reaction optimisation, involving the addition of dNTPs and MgCl2. The presence of PICs provided information on the amplification performance and post-PCR processing and can assist with the interpretation of human DNA profiles.
The position of the fragments also allowed PICs to be used as sizing standard. Compared to the GSTh 500 ROXTM size standard, PICs showed a slightly lower sizing precision, with standard deviations lower than 0.3 bp. Even though this resulted in allelic bins higher than 1 bp limit for 99.7% confidence, all samples sized with PICs were correctly genotyped.
The addition of PICs would be a valuable tool, in particular, for the analysis of compromised DNA. In particular it would be useful when analysing DNA recovered from skeletal remains, which are prone to accumulation of PCR inhibitors and DNA degradation.

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