AmpFlSTR Profiler Plus Research Articles

Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs.

The primary method for analysis of low template DNA (LTDNA) is known as the low copy number (LCN) method involving an increased number of PCR cycles (typically 34). In common with other LTDNA methods, LCN profiles are characterized by allelic imbalance, drop in, and drop out that require complicated interpretation rules. They often require replicate PCR reactions to generate a "consensus" profile in a specialized facility. An ideal method for analysis of LTDNA should enhance profiling outcomes without elevated error rates and be performed using standard facilities, with minimum additional cost. In this study, we present a comparison of four method variations for the amplification of STRs from LTDNA with a widely used, commercially available kit (AmpFℓSTR(®) Profiler Plus(®)): the standard method, the standard method with a post-PCR clean up, the LCN method, and a reduced reaction volume with increased Taq DNA polymerase concentration. Using telogen hairs-a common source of LTDNA-and matched reference DNA, the LCN method produced the highest number of concordant and non-concordant (i.e., dropped-in) alleles. In comparison, the reduced reaction volume with increased Taq polymerase yielded more full and concordant DNA profiles (all alleles combined) and less off-ladder alleles from a broad range of input DNA. In addition, this method resulted in less non-concordant alleles than LCN and no more than for standard PCR, which suggests that it may be preferred over increased PCR cycles for LTDNA analysis, either with or without consensus profiling and statistical modelling. Overall, this study highlights the importance and benefit of optimizing PCR conditions and developing improved laboratory methods to amplify and analyze LTDNA.

Screening of longevity-associated genes based on a comparison between dead and surviving populations

AimTo investigate longevity-associated genes based on a comparison between dead and surviving populations. MethodsA total of 71 cases of dead individuals were treated as the death group, and healthy volunteers who were matched with the dead individuals based on sex and age were recruited as the survival group. Alleles of 13 CODIS STR loci were determined using the AmpFLSTR Profiler Plus PCR Amplification Kit. The cross-validation was performed based on differences between the two groups in both frequency values and ages. ResultsThe frequency value of the D18S51-17 alleles was significantly higher in the dead group than in the survival group (p<0.05), and the frequency value of the D2S1338-18 allele was statistically lower in the dead group than in the survival group (p<0.05). The mean age of the subjects with the D2S1338-18 allele was also significantly higher than that of the subjects without D2S1338-18, and no significant difference was observed with respect to the other three alleles. ConclusionsThe results suggest that D2S1338-18 is associated with longevity.

Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing

We sequenced the D21S11 locus in 77 individuals from Danish paternity cases using 454 FLX next generation sequencing (NGS) technology. All samples were also typed with the AmpFlSTR Profiler Plus or the AmpFlSTR Identifiler PCR Amplification kits as part of paternity investigations. In 18 of the confirmed trios, a genetic inconsistency was observed between one of the parents and the child at the D21S11 locus. NGS of the D21S11 locus revealed which allele had mutated from which parent to the child in 13 of these trios. All characterized mutations could be explained by single-step mutations in the longest sub-repeat of D21S11. A total of 53 of the 77 sequenced samples originated from unrelated individuals. Twenty different D21S11 alleles were detected by NGS in these individuals whereas only 13 different alleles were observed with fragment analysis. Several alleles had the same lengths but different sequences, e.g. four and three different alleles were detected by NGS with lengths determined by CE corresponding to allele 30 and allele 31, respectively.

Sperm Hy-Liter™: An effective tool for the detection of spermatozoa in sexual assault exhibits

A fluorescence-based assay specifically targeting human spermatozoa was tested and optimized for best staining results using a variety of mock sexual assault samples. Swab clippings versus whole swabs were evaluated for best sample preparation and to simplify workflow (direct application versus swab extraction). The practicality and sensitivity of Sperm Hy-Liter™ was compared to our current phase contrast microscopy protocol for searching for the presence of spermatozoa. Sperm Hy-Liter™ was more sensitive than phase contrast microscopy and was able to detect spermatozoa more effectively in actual sexual assault samples (recent [N=240] or 24 years old [N=4]) containing few spermatozoa. Correlations were drawn between the Sperm Hy-Liter™ spermatozoa counts and the AmpFlSTR(®) Profiler(®) Plus male profiles generated from the sperm cell DNA fractions of semen containing swabs and swab clippings. In addition, recovered spermatozoa from Sperm Hy-Liter™-stained slides with greater than 40 spermatozoa produced full STR male profiles in 20.3% of slides tested and partial STR male profiles in 52.8% of slides tested. The adoption of Sperm Hy-Liter™ offers a means to standardize and improve the efficiency of the microscopic screening of sexual assault evidence.

Intrahepatic recurrence of hepatocellular carcinoma 13 years after orthotopic liver transplantation for hepatitis C-related cirrhosis with occult hepatocellular carcinoma: A case report

Intrahepatic recurrence of hepatocellular carcinoma 13 years after orthotopic liver transplantation for hepatitis C-related cirrhosis with occult hepatocellular carcinoma: A case report

A Rapid Wire‐Based Sampling Method for DNA Profiling*

This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)-based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser(TM). Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom-designed quintuplex primer set suitable for use with the Bioanalyser(TM). Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR(®) Profiler Plus(®) without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required.

Monozygotic twins with variable expression of Van der Woude Syndrome

Monozygotic twins with variable expression of Van der Woude Syndrome

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow-up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpFlSTR COfiler and AmpFlSTR Profiler Plus ID kits. Results showed that heat treatments via microwave or Biz/Na(2)CO(3) in sub-boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long-term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.

DNA profiles from flip-open cell phones

Flip-open style cell phones were investigated for the potential to produce quality genetic profiles that could be used in forensic casework. Swabs were taken of the outside/back and the inside ear speaker of ten flip-phones on two occasions – prior to and seven days after cleaning with 95% ethanol. Buccal swabs were collected as exemplars. The samples were amplified using the AmpFlSTR ProfilerPlus PCR Kit for 35 cycles and STR profiles were generated using an ABI Prism 310 Genetic Analyzer and GeneMapper ID analysis software v3.2. The phone profiles were compared to the references and to each other, to assess the quality of the profiles. The completeness of the profiles varied greatly, even within an experimental condition. There was no significant difference in the percentage correct alleles or in the number of drop-in alleles in the DNA profiles for the outside/inside locations or for the pre/post-cleaning times. The findings of this study demonstrate the need for collecting multiple samples from discrete locations on a cell phone if such evidence is encountered in forensic cases.

Systematic analysis of interference due to stutter in estimating chimerism following hematopoietic cell transplantation

Stutter formed during amplification of short tandem repeats (STRs) interfere with accurate engraftment monitoring following hematopoietic cell transplantation (HCT). We describe a mathematical approach to minimize the contribution of stutter when estimating chimerism following HCT. Pretransplant DNA samples from 409 donors and recipients were used to define marker-specific stutter cut-off values for all makers used in the AMPFlSTR Profiler Plus amplification kit. Mock chimerism samples (5, 20, and 50%) were used to evaluate the contribution of stutter in estimating chimerism. Three markers, vWA, D13S317, and D18S51, had overlapping stutter in the mock chimerism samples. Only D18S51 had stutter from a shared allele, whereas the other two markers had stutter from nonshared alleles. Without adjustment for stutter, D18S51 showed a 8, 6, and 4% difference from expected chimerism values (5, 20, and 50%) and after adjusting for laboratory-defined marker-specific stutter cut-offs the corresponding difference from expected chimerism values was 1, 0, and 0%. Difference from expected chimerism values in the vWa and D13S317 markers were similar before and after adjustment of stutter. Adjustment for stutter from shared alleles may improve accuracy of estimated chimerism.

Developing STR databases on structured populations: The native South Siberian population versus the Russian population

Developing a forensic DNA database on a population that consists of local ethnic groups separated by physical and cultural barriers is questionable as it can be genetically subdivided. On the other side, small sizes of ethnic groups, especially in alpine regions where they are sub-structured further into small villages, prevent collecting a large sample from each ethnic group. For such situations, we suggest to obtain both a total population database on allele frequencies across ethnic groups and a list of θ-values between the groups and the total data. We have genotyped 558 individuals from the native population of South Siberia, consisting of nine ethnic groups, at 17 autosomal STR loci of the kit packages AmpF lSTR SGM Plus и AmpF lSTR Profiler Plus. The groups differentiate from each other with average θ-values of around 1.1%, and some reach up to three to four percent at certain loci. There exists between-village differentiation as well. Therefore, a database for the population of South Siberia is composed of data on allele frequencies in the pool of ethnic groups and data on θ-values that indicate variation in allele frequencies across the groups. Comparison to additional data on northeastern Asia (the Chukchi and Koryak) shows that differentiation in allele frequencies among small groups that are separated by large geographic distance can be even greater. In contrast, populations of Russians that live in large cities of the European part of Russia are homogeneous in allele frequencies, despite large geographic distance between them, and thus can be described by a database on allele frequencies alone, without any specific information on θ-values.

The variation of 15 autosomal microsatellite DNA loci in five indigenous populations of South Siberia

The allele frequencies of 15 autosomal STR loci (D3S1358, vWA , FGA , TH01 , TPOX , CSF1PO , D5S818, D13S317, D7S820, D16S539, D2S1338, D8S1179, D21S11, D18S51, and D19S433) included into the AmpFlSTR SGM Plus and AmpFlSTR Profiler Plus kits (Applied Biosystems, United States) were deter- mined for five indigenous populations of South Siberia: Buryats, Altaians, Tofalars, Sojots, and Khakassians ( N = 261). No significant differences in allele frequencies were found between the populations. The combined power of discrimination of the STR loci was determined for every population. DOI: 10.1134/S0026893307040024

A Single Mutation in the FGA Locus Responsible for False Homozygosities and Discrepancies Between Commercial Kits in an Unusual Paternity Test Case

We report an unusual paternity test case showing multiple peculiarities. Using AmpFlSTR Profiler Plus and AmpFlSTR Identifiler PCR Amplification kits, the alleged father and the two children were apparently homozygous at the FGA locus, but using the PowerPlex 16 kit the three individuals were found to be heterozygous. Drop-out was caused by a single mutation event in the presumptive binding site of the reverse primer. In addition, three inconsistencies were detected between the daughter and the alleged father among 18 STR markers. The occurrence of the rare null allele at the FGA locus and case history suggested that the true father was the brother of the alleged father. Furthermore, a single-step repeat maternal mutation was also detected at D16S539. This puzzling case was solved by using multiple analytical approaches, including the use of different primer pairs, the use of a high number of STR markers, and the characterization of the mutation causing the "null allele."

Rapid and High‐Throughput Forensic Short Tandem Repeat Typing Using a 96‐Lane Microfabricated Capillary Array Electrophoresis Microdevice*

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.

Detection of non-maternal components of gestational choriocarcinoma by PCR-based microsatellite DNA assay

Objective. Gestational and non-gestational choriocarcinomas have distinctly different tissues of origin, parental genotypes, natural histories, and responses to therapy. Our objective was to develop a convenient, fast, and reliable assay that would, using only patient tissue, allow separation of gestational from non-gestational choriocarcinomas. Method. Benign and malignant tissues, preserved in paraffin blocks and separated by microdissection, were examined using a commercial PCR-based tissue identity assay (ABI AmpFlSTR Profiler Plus Kit and ABI 377 DNA sequencer) to detect genetic profiles of 9 microsatellite markers, along with X and Y chromosome markers. Cases included 6 choriocarcinomas. Controls included eight non-germ cell reproductive tract tumors and two hydatidiform moles. Results. The microsatellite markers identified the five choriocarcinomas diagnosed on clinical and histological grounds as gestational, to be of genetically non-maternal (androgenic) origin. The neoplasm previously classified as a non-gestational choriocarcinoma was demonstrated to be of maternal origin, as were the non-germ cell reproductive tract tumors. Samples from hydatidiform moles contained either androgenic markers only or a mix of maternal and androgenic markers, as previously seen in complete and partial moles, respectively. Conclusion. A commercially available microsatellite DNA diagnostic assay is a quick and convenient way to discriminate between gestational and non-gestational choriocarcinoma.

Beware of the Possibility of Fingerprinting Techniques Transferring DNA

Fingerprinting brushes have the potential to collect and transfer DNA during powdering. Squirrel-hair fingerprint brushes exposed to specific sets of saliva stains and brushes used in routine casework were tested for their ability to collect and transfer DNA containing material using standard DNA extraction procedures and AmpFlSTR Profiler Plus amplification and typing procedures. The tests found that the risk of transferring DNA during powdering and having a detrimental impact on the analysis increases if the examiner powders over either biological stains (such as blood or saliva) or very fresh prints and uses more sensitive PCR amplification and typing procedures. We advocate caution when powdering prints from which DNA may also be collected and provide options for consideration to limit the risk of transferred DNA contamination while fingerprinting.

STR Data for the AmpFℓSTR Profiler Plus from Western China

Blood samples were collected from unrelated individuals ofChinese Han ethnic group in Chengdu of China. DNA was ex-tracted using Chelex method (1). 0.5–2ng target DNA followedthe protocols described in the AmpFlSTR Cofiler user’s manual(PE Applied Biosystems), the exception was that the PCR vol-umewas25µL.ThesampleswereamplifiedusingGeneAmpPCRSystem 9700 (Applied Biosystems, Foster City, CA). DNA typ-ing was carried out on the ABI 3100 Genetic Analyzer (AppliedBiosystems). The amplified products were detected with the ABIPRISM 3100 Genetic Analyzer using the separation medium Per-formance Optimized Polymer (POP) 4

Microsatellite Diversity in Andhra Pradesh, India: Genetic Stratification Versus Social Stratification

DNA samples of 948 individuals belonging to 27 populations from southern Andhra Pradesh were analyzed for nine AmpFlSTR Profiler Plus loci. The nature and extent of genomic diversity within and between these populations have been examined with reference to socioeconomic and geographic affiliations. The results suggest that the average heterozygosity is uniformly high in these populations (> 0.80) and that the patterns of allele distributions are similar across the populations. The value of the coefficient of gene differentiation and the AMOVA and structure analysis results suggest that these populations are highly homogeneous. The neighbor-joining tree constructed using either D(A) or F(ST) distances suggests no intelligible pattern of population clusters based on ethnohistoric or geographic affiliations. All these observations suggest either a common recent origin of these populations or extensive gene flow across the populations that erased the original genetic differences. Given strict endogamy, the latter explanation can hold only if there has been unauthorized or unrecognized gene flow transecting the social boundaries. Nevertheless, the regression plot of average heterozygosity versus distance from the centroid (Rii), based on Harpending and Ward's (1982) model, and the genetic distances computed between different hierarchical groups within Andhra Pradesh tend to support this conjecture. Overall, the results suggest lack of a significant degree of genetic stratification that is consistent with social stratification in Andhra Pradesh. Furthermore, the neighbor-joining tree based on comparative data from other Indian and continental populations brings out a single and compact cluster of all the Andhra populations that is clearly separated from the rest.

Genetic polymorphisms at 13 STR loci in autochthonous Basques from the province of Alava (Spain)

In this study, allelic frequencies of the CODIS core short tandem repeat (STR) loci were estimated in a population sample consisting in 101 unrelated healthy autochthonous individuals from the Basque province of Alava (Northern Spain). Frequency distributions for all 13 STR loci were obtained using the AmpF lSTR Profiler Plus and AmpF lSTR COfiler amplification kits. The allelic frequencies generated were employed to calculate genetic and forensic useful parameters.

Systematic Analysis of Stutter Percentages and Allele Peak Height and Peak Area Ratios at Heterozygous STR Loci for Forensic Casework and Database Samples

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpFlSTR Profiler Plus (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 +/- 6.5% for database samples, 88 +/- 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median -3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.