A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Verheij, Saskia; Harteveld, Joyce; Sijen, Titia

doi:10.1016/j.fsigen.2011.03.014

Cited by 73 publications

(60 citation statements)

References 14 publications

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“…Fast PCR and/or direct PCR are fairly recent improvements from the late 2000s/early 2010s that can be utilized by the forensic DNA community to reduce processing time and/or costs for forensic-type and reference samples [1][2][3][4][5][6][7][8][9][10]. Reduced volume PCR amplification has been shown to improve sensitivity and efficiency [11], but the more reduced the total reaction volume becomes, the more intra-locus peak balance decreases, making extremely low volume reactions likely unsuitable for anything other than single-source reference samples.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of Four Polymerases for Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Connon¹

2016

FLIS

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Section: Introductionmentioning

confidence: 99%

“…Using fast thermal cyclers, fast PCR has been achieved in as little as 19-26 min [1,2,5]. In general, fast PCR profiles have often suffered from low level artifacts, increased n-4 stutter percentage and incomplete adenylation (-A) [1,2,5,[8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Assessment of Four Polymerases for Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Connon¹

2016

FLIS

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“…Maximum stutter limits per locus were calculated via the sum of average percent stutter plus three standard deviations (a more conservative approach adds two standard deviations [11]), which never exceeded 20% for any locus or amplification method. It should be noted, however, that the maximum observed percent stutter (n-4) was occasionally (0.05-0.18% of samples) above 20% for all Identifiler and Identifiler Plus fast PCR methods, ranging from 21-25%, but never exceeded 20% for PowerPlex 16 HS.…”

Section: Determination Of the Optimal Range Of Input Dnamentioning

confidence: 99%

“…Reducing amplification processing time has been a major focus for forensic DNA testing over the past 10 years, especially through the advent of fast PCR polymerases, direct PCR, and rapid DNA testing [1][2][3][4][5][6][7][8][9][10][11][12]. But such a reduction in processing time has often been subject to an increase in cost due to reagents and/or equipment, and/or prone to a decrease in STR profile quality.…”

Section: Introductionmentioning

confidence: 99%

Validation of Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Connon¹

2016

FLIS

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This project was a continuation of a previous project that examined various polymerases to assess their suitability for low volume, fast PCR amplification of forensic STR loci. The primary focus of this project is to develop multiple protocols using the product selected from the previous study (KAPA2G™ Fast Multiplex PCR Kit) along with primer sets and non-fast thermal cyclers commonly used in the forensic community in order to substantially reduce PCR time without added costs or equipment. Ultimately, 3µl fast PCR protocols were validated for the PowerPlex® 16 HS System, AmpFℓSTR® Identifiler® and Identifiler® Plus PCR Amplification Kit primer sets on a 384-well Veriti® thermal cycler (43-51min), as well as 5μl and 6μl Identifiler® fast reactions on a GeneAmp® PCR System 9700 thermal cycler (51min). Two-step PCR cycling utilizing a combined annealing/extension step was successfully incorporated into the fast PCR protocols for all primer sets, except PowerPlex® 16 HS. These protocols were validated for use with forensic reference-type samples, including buccal swabs or Buccal DNA Collectors™, based on optimal DNA input range (sensitivity, reproducibility, inter-and intra-locus peak balance, stutter, pull-up, incomplete adenylation (-A), and baseline assessment), stochastic, precision, stutter, automation, contamination, lot-to-lot variation and storage condition studies. Overall, a 56-73% reduction in amplification time was achieved compared to low volume, standard PCR amplification, along with STR profile pass rates of 95-98% (n=86-89) using these low volume, fast PCR protocols, all of which had an optimal DNA input range of 0.375-1.5ng. Higher percent stutter was observed compared to that of standard PCR, but non-specific amplification, incomplete non-template adenylation, and poor intra-locus peak balance were not problematic compared to other previous studies involving fast PCR.

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“…Recently, a 6-h DNA typing service has been developed and validated by the NFI [16]. This has been achieved by integrating a fast PCR protocol with an inhibitor tolerant DNA polymerase.…”

Section: Integrated Forensic Platform Projects At the Netherlands Formentioning

confidence: 99%

The interface between forensic science and technology: how technology could cause a paradigm shift in the role of forensic institutes in the criminal justice system

Kloosterman

Mapes

Geradts

et al. 2015

Phil. Trans. R. Soc. B

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In this paper, the importance of modern technology in forensic investigations is discussed. Recent technological developments are creating new possibilities to perform robust scientific measurements and studies outside the controlled laboratory environment. The benefits of real-time, on-site forensic investigations are manifold and such technology has the potential to strongly increase the speed and efficacy of the criminal justice system. However, such benefits are only realized when quality can be guaranteed at all times and findings can be used as forensic evidence in court. At the Netherlands Forensic Institute, innovation efforts are currently undertaken to develop integrated forensic platform solutions that allow for the forensic investigation of human biological traces, the chemical identification of illicit drugs and the study of large amounts of digital evidence. These platforms enable field investigations, yield robust and validated evidence and allow for forensic intelligence and targeted use of expert capacity at the forensic institutes. This technological revolution in forensic science could ultimately lead to a paradigm shift in which a new role of the forensic expert emerges as developer and custodian of integrated forensic platforms.

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A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Cited by 73 publications

References 14 publications

Assessment of Four Polymerases for Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Assessment of Four Polymerases for Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Validation of Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

The interface between forensic science and technology: how technology could cause a paradigm shift in the role of forensic institutes in the criminal justice system

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