Demonstration of rapid multiplex PCR amplification involving 16 genetic loci

National Institute of Standards and Technology, Biochemical Science Division, 100 Bureau Drive, Mail Stop 8311, Gaithersburg, MD 20899-8311, USA.
Forensic Science International: Genetics (Impact Factor: 4.6). 01/2009; 3(1):42-5. DOI: 10.1016/j.fsigen.2008.09.005
Source: PubMed


Current forensic DNA typing is conducted in approximately 8-10h. Steps include DNA extraction, quantification, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection, data analysis and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately 3h with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 min. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. Complete concordance of STR allele calls (for 60 samples) between the rapid and standard thermal cycling protocols were observed although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 750 pg of template DNA and 28 cycles, STR peaks for all loci were above a 150 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.84.

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    • "The loci amplified by this kit includes the 13 Combined DNA Index System (CODIS) core STRs loci, two additional widely used STRs (D2S1338 and D19S433) and the sex-marker Amelogenin [12]. Fast PCR protocols have been established by other research groups for multiplex amplification of STRs, including Identifiler; however, each group experienced their own set of challenges [2-5]. "
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    ABSTRACT: Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.
    Full-text · Article · Mar 2012 · Investigative Genetics
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    • "According to the Tsukada et al. [1] experiment in order to reduce requiring time for amplification work, the processing time can be reduced down to 36 min when using new buffer and new enzyme. Also, Vallone et al. [2] have experiment rapid PCR by using newly developed PyroStart (Fermentas, Glen Burnie, MD) and SpeedSTAR (Takara Bio USA, Madison, WI). Thus, the purpose of this study is to establish amplification conditions with reproducibility and reliability and less time consumption by using existing equipments and reagent without changing buffer and polymerase. "
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    ABSTRACT: Current forensic DNA typing is conducted in approximately 8–9 h. Whole steps included DNA extraction step, Quantification, PCR amplification step, electrophoresis process through capillary separation with fluorescence detection, data analysis and DNA profile interpretation. Among them, we have tested rapid PCR method of AmpFlSTR Identifler PCR to reduce running time. We have altered several PCR conditions of ordinary AmpFlSTR Identifler PCR method and used 9947A control DNA to cut the time for the PCR reaction. In the results of this study, the critical step of the PCR reaction was the annealing step and also we reduced PCR running times by 1/3 to 2/3 (approximately 60–90 min) with complete concordance of STR allele calls using standard reference material 9947A.
    Full-text · Article · Dec 2011 · Forensic Science International Genetics Supplement Series
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    • "All the fast short-fragment PCR assays were more sensitive than the TaqMan ® assays (data not shown). Vallone et al. (2008) demonstrated that the highly processive SpeedSTAR TM HS polymerase (∼100 nucleotides/s) could amplify 300 bp fragments in 10 ␮l reaction volumes using extension times of 10 s only. Using the five previously designed primer sets for the detection of WSSV, IHNNV, and MBV viruses by TaqMan ® real-time PCR (Table 1), the fast short-fragment PCR was more sensitive than TaqMan ® assays. "
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    ABSTRACT: This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 degrees C denaturation and 60 degrees C annealing/extension) PCRs were performed in 5-10 microl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25-30 min. Rapid high-resolution agarose gel electrophoresis of 70-150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50-100 pg) was obtained using ultra sensitive dyes such as GelStar and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan real-time PCR counterparts.
    Full-text · Article · Sep 2010 · Journal of virological methods
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