Lori K. Hennessy

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

Development and Validation of the AmpFℓSTR® Yfiler™ PCR Amplification Kit: A Male Specific, Single Amplification 17 Y-STR Multiplex System*

ABSTRACT: In the past 5 years, there has been a substantial increase in the use of Y‐short tandem repeat loci (Y‐STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFℓSTR® Yfiler™ PCR amplification kit simultaneously amplifies 17 Y‐STR loci including the loci in the “European minimal haplotype” (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y‐STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler™ kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross‐reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.

Development and Validation of the AmpFℓSTR® MiniFilerTM PCR Amplification Kit: A MiniSTR Multiplex for the Analysis of Degraded and/or PCR Inhibited DNA

Abstract:  DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFℓSTR® MiniFiler™ PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFℓSTR® Identifiler® PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFℓSTR® SGM Plus® kit. This informative nine‐locus multiplex, which includes the gender‐identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFℓSTR® Identifiler® and SGM Plus® kits. These data support that the MiniFiler™ kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.

Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples.

The Quantifiler Human DNA Quantification Kit and the Quantifiler Y Human Male DNA Quantification Kit were designed for the quantification of human genomic DNA in forensic samples. The kits use a real-time PCR-based process to quantify, respectively, total human DNA or human male DNA only. We report the results of a developmental validation study that we performed with the Quantifiler Kits, following the official SWGDAM guidelines. The Quantifiler Kits were tested for performance criteria such as species specificity, sensitivity, stability, precision and accuracy, and in addition, were tested with forensic case-type samples and mixed (male:female) DNA samples. The Quantifiler Kit methods were highly specific for human DNA, and could detect as little as 32 picograms of DNA using 2 microL of sample per assay. The accuracy and precision of the Quantifiler Kit methods was comparable or superior to that of other quantification methods.

Null allele sequence structure at the DYS448 locus and implications for profile interpretation.

Null alleles can occur with any PCR-based STR typing system. They generally are due to deletions within the target region or primer binding sites or by primer binding site mutations that destabilize hybridization of at least one of the primers flanking the target region. Although not common, null types were detected at the DYS448 locus in seven out of 1,005 unrelated males in the Hispanic population. Of these DYS448 null types, four individuals displayed an apparent duplication at the DYS437 locus. The additional allele observed at the DYS437 locus is in actuality a smaller-sized DYS448 amplicon, which is the result of a deletion of the invariant N42 base pair domain and downstream repeats within the DYS448 locus. Thus, some DYS448 null types are not truly null. A true DYS448 null allele carried numerous primer binding site variants and a large deletion including the N42 base pair domain and surrounding or downstream repeat regions. The presence of null alleles is not a real concern for interpretation of Y STR loci evidence; current methods for interpreting Y STR profiles easily accommodate such phenomena.

Developmental validation of the GlobalFiler® express kit, a 24-marker STR assay, on the RapidHIT® System

Rapid DNA typing provides a transformative solution to help forensic laboratories and law enforcement agencies solve and prevent crimes. The RapidHIT(®) System is a fully integrated instrument with a simplified user interface enabling an operator to run the system and obtain a DNA profile from a sample in less than two hours. The integration and developmental validation of the NDIS-approved 24 loci GlobalFiler(®) Express kit expands the capabilities of the RapidHIT System to increase discrimination power, reduce adventitious matches, and improve cross-border data sharing capabilities. Developmental validation studies were performed according to the SWGDAM guidelines and tested several critical areas of performance including three sensitivity studies, inhibited samples, thermal cycling parameters, and cross-contamination. Validation studies indicate that the optimized PCR parameters and sensitivity of the system is capable of generating STR profiles from buccal or blood swab reference samples. Results were concordant with genotypes produced using standard bench thermal cyclers and capillary electrophoresis platforms. Furthermore, swabs can be retrieved from the system and re-run or reprocessed with traditional bench chemistries, e.g. Y-STRs, to gain additional information. Our results demonstrate that the GlobalFiler Express assay run on the RapidHIT System is reliable for generating profiles from reference samples after forensic review.

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples.

UNLABELLED Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT® System, capable of producing up to five high quality STR profiles with full controls in approximately 90min using PowerPlex®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis. The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform. RESULT are concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.

Characterization of the N+3 Stutter Product in the Trinucleotide Repeat Locus DYS392

ABSTRACT: Stutter products generated during DNA amplification by the polymerase chain reaction (PCR) may complicate mixture interpretation. The PCR amplification of the DYS392 locus typically results in three distinct detectable PCR products: the true allele product (N), a stutter product three bases smaller (N−3), and a reproducible low‐level product, three bases larger (N+3). Sequence analysis of the N+3 product demonstrated that its sequence is one TAT repeat longer than the true allele product. Our experiments demonstrated that the quantity of both N−3 and N+3 stutter increased as the allele number increased. The percent stutter also increased as the magnesium concentration was increased in the reaction, as well as when the amount of input DNA was decreased. As both stutter products behave in a similar and reproducible fashion, the same rules that apply to the interpretation of N−3 stutter products in short tandem repeat analysis, can be applied to N+3 stutters. The characterization of the DYS392 N+3 product is the first detailed published study of a stutter product larger than the true allele.

Applications of 5-dye technology in forensic DNA typing and analysis

In the past decade, fluorescence-based DNA detection systems have been widely used in forensic DNA analysis. Fluorescence detection methods have greatly aided the sensitivity and ease of measurement of PCR amplified short tandem repeat (STR) alleles. In the new multiplexed STR genotyping kits, fluorescent dyes are covalently coupled to primers for each locus. Fluorescence measurements involve detecting light emitted from an excited dye at a specific wavelength. Filters are used to select fluorescent dye signals at optimal wavelength ranges. In ABI PRISMR instruments, fluorescent signals are separated by a diffraction grating and projected onto a chargecoupled device (CCD) camera during data collection. Multicomponent analysis is performed using a matrix that excludes the contribution of neighboring dyes. Earlier kits from Applied Biosytems (AmpFlSTRRkits) used PCR primers with NHS-ester dyes 5-FAMk, JOEk or NEDk and ROXk dyes and emission spectra ranging from 522 to 607 nm. More recently, Applied Biosystems has developed a unique 5-dye technology for automated DNA fragment analysis. The introduction of the new 5-dye chemistry involves replacement of 5-FAM with 6-FAMk, JOE with VICk and ROX with LIZk and incorporation of the new PETk dye into the existing system. The five dyes, 6-FAM, VIC, NED, PET and LIZ, expand the spectral detection range on the ABI PRISM instrumentation to 660 nm. The addition of more dyes thus enables more loci to be multiplexed into a single PCR amplification. Additionally, the new dyes will increase genotyping throughput significantly since more loci can be analyzed simultaneously in a single lane or capillary. One advantage of this arrangement is that minimal hardware changes to existing instrument platforms is necessary.

Identification of a Novel Polymorphism in the X‐Chromosome Region Homologous to the DYS456 Locus

ABSTRACT: During an extensive multipopulation study with Y‐short tandem repeat (STR) loci, amplified using the AmpFℓSTR® Yfiler™ PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N=3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single‐nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X–Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub‐Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y‐STR loci typeable using the AmpFℓSTR® Yfiler™ PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFℓSTR Yfiler kit even in the presence of female DNA containing the G variant.