Research article
Rapid STR analysis of single source DNA samples in 2 h

https://doi.org/10.1016/j.fsigss.2009.08.070Get rights and content

Abstract

The establishment of offender DNA databases is critical to future crime prevention. Many countries have established databases or are in the process of passing database legislation. With new legislation the number of samples that will be collected could begin to exceed the testing capacity of many labs leading to backlogs.

Two bottlenecks in the workflow that can contribute to a backlog of samples are DNA purification and PCR cycling time. The average purification time is approximately 2 h and the average cycling time of current STR kits is approximately 3 h. To address the second problem we investigated alternative DNA enzymes to decrease PCR cycling time. It was necessary to balance the increase in time to result against the need to address factors which can impact interpretation of a DNA profile such as: generation of stutter products, non-template addition, intra-locus balance, accuracy, and species specificity.

Initial feasibility studies demonstrate that alternative enzymes can decrease PCR cycling time. The data show that this assay can increase throughput, providing results in less than 2 h. However, decreasing PCR cycling time will have an affect on multiplex STR performance.

Introduction

Criminal DNA databases have been established in many countries around the world. The underlying concept behind the creation of a national DNA database of convicted offender profiles was to use it to solve crimes where there are no known suspects. To keep up with existing DNA databasing demands, as well as prepare for current and future legislative database expansion, DNA laboratories are looking to reduce time to result, increase throughput, and minimize the manual steps in the workflow. Current PCR cycling times are around 2.5–3 h. Decreasing the time during this step in the workflow would increase throughput. Previous studies have shown that alternative enzymes can decrease cycling time [1]. To decrease PCR cycling time we have screened alternative enzymes for the ability to decrease PCR cycling time.

Section snippets

Materials and methods

The AmpFℓSTR® SGM Plus® or Identifiler® PCR Amplification Kit primer sets were used for all experiments [2]. The different enzymes tested were: AmpliTaq Gold®, Phusion (NEB), AB77, AB95, AB-1, and AB-3 (Applied Biosystems, LLC).

The PCR amplifications were performed with 1 ng of control DNA 007 in a reaction volume of 25 μl with MicroAmp reaction tubes (Applied Biosystems) in the GeneAmp PCR Systems 9700 with a gold-plated silver or silver block or the Veriti® thermal cycler (Applied Biosystems).

Results and discussion

Thermal cycling times were decreased to approximately 46 min using the Veriti® thermal cycler but the Phusion, AB77 and AB95 enzymes generated non-specific amplification products compared to the AmpliTaq Gold® enzyme (data not shown). This was probably caused by non-specific priming during assay set-up as these were not hot start enzymes. The AB-3 enzyme generated a clean profile but contained extra n + 1 and n  2 stutter peaks. Of the five alternative enzymes, AB-1 generated the cleanest DNA

Conclusion

The preliminary results demonstrate that AB-1 enzyme can be used to increase throughput for single source database samples. The enzyme decreases PCR cycling to under 1 h. Other experiments (not shown here) have demonstrated that sensitivity and intralocus balance were comparable to our current Identifiler and SGM Plus STR kits.

However, the alternative enzyme yields higher stutter peaks and higher species specific cross reactivity. Additional experiments are ongoing to further improve the assay.

Conflict of interest

None.

Role of funding

This research was funded by Applied Biosystems, a part of Life Technologies. The authors are employees of the company.

Acknowledgements

Lisa M Calandro.

References (2)

Cited by (20)

  • Forensic STR profiling based smart barcode, a highly efficient and cost effective human identification system

    2018, Saudi Journal of Biological Sciences
    Citation Excerpt :

    Some of rapid methods have already been published and utilization of such methods to reduce and time is gaining momentum (Azari et al., 2007; Wang et al., 2009). These efficient techniques were merged with technology to maintain the criminal DNA databases at national and international level (Wang et al., 2009) as well as the establishment of new technological methods for human identification like human barcode and RFID chip as an identifier within limited range. These conventional techniques including a microchip that can transmit a static identifier has many potential socioeconomic and health hazards.

  • Validation study of a 15-plex rapid STR amplification system for human identification

    2017, Forensic Science International: Genetics
    Citation Excerpt :

    The observed stutter ratio for this rapid PCR system was on average 13.68% compared to the average of 10% from the recommended filtering threshold for the standard DNATyper™ 15 amplification system [21]. The augmentation of stutter percentages has been reported by other rapid PCR protocols, so it is not unexpected [5,9,12,15,20,24]. Using the same rapid amplification of AmpFlSTR® Profiler Plus, both the Laurin and Foster groups report stutter percentage values increasing by 2.2% and 2–3%, respectively [5,24].

  • Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples

    2016, Forensic Science International: Genetics
    Citation Excerpt :

    As technology continues to advance, there is a certain expectation that such advancements will result in a decrease in processing time and cost. Previous studies have demonstrated the ability to reduce processing time via fast PCR and/or direct amplification [1–12], while capillary electrophoresis detection time can be reduced using a combination of POP-6™ Polymer (POP-6; Applied Biosystems, Foster City, CA) and a 22 cm array [13] or using a 3500 series Genetic Analyzer (Applied Biosystems) [14]. Rapid DNA testing has also been achieved in less than two hours from start to finish using a single instrument, and has demonstrated its suitability for investigative leads when time is a crucial factor [15,16], though it is not a cost effective means of testing for high-throughput laboratories.

  • Rapid PCR of STR markers: Applications to human identification

    2015, Forensic Science International: Genetics
    Citation Excerpt :

    Further work added Premix Ex Taq to the cocktail previously optimized for rapid thermal cycling of the IdentiFiler chemistry and reduced all occurrences of the previously observed incomplete adenylation and artifacts, while maintaining the rapid speed of amplification [21]. Five unique alternate polymerases in addition to AmpliTaq Gold (Phusion, AB77, AB95, AB-1, and AB-3) were tested with a rapid PCR protocol and generated full STR profiles in 46 min with the Veriti thermal cycler [22]. In this work, Phusion, AB77 and AB95 all produced PCR products which were non-adenylated and thus one basepair short of the commercial allelic ladder for the IdentiFiler and SGM Plus kits.

  • Development and evaluation of a rapid PCR method for the PowerPlex®S5 system for forensic DNA profiling

    2014, Legal Medicine
    Citation Excerpt :

    Various technologies that decrease the time required for DNA typing have been developed including microfluidic biochips [1–3] and continuous flow micro channels [4], and most recently platforms that perform the entire DNA typing process in a single instrument in approximately 90 min [5–6]. Another approach has been the development of faster chemistries and the use of alternate polymerases [7–13]. Fast chemistry involves the application of new polymerases with higher extension rates and decreased activation times but maintaining the levels of sensitivity and fidelity of standard polymerases.

View all citing articles on Scopus
View full text