NGM SElectTM Kit Research Articles

Effect of the activity in secondary transfer of DNA profiles

Secondary transfer occurs when DNA is transferred from one object, surface or person to another, via an intermediate object/person [1].Currently, some lawyers are using the existence of this type of transfer as an argument to invalidate genetic analysis. In a recent case in Spain, the existence of secondary transfer was alleged from part of the defense to justify the presence of DNA of suspect on a specific object found in the crime scene.In the present work, an attempt has been made to confirm the presence of a person's DNA in an object, via an intermediate person. To test the hypothesis we used individuals who are very often in contact, such as couples. It is intended to assess if, in those cases, it is possible to obtain a genetic profile after performing a current activity such as washing hands and dry with a towel. Two experiments were performed. First, the primary transfer was evaluated. We asked 10 people (5 men and 5 women) with different daily activities, to wash their hands with neutral pH soap, and then dry their hands with a specific towel.In the second experiment, the secondary transfer was evaluated. Five couples were asked to hold their hands for 5 min. Then, they were asked to wash their hands with neutral pH soap, and dry them on a different towel each. This experiment was performed 3 times each pair. As a control, an unused piece of each towel was analysed, and saliva samples were taken from all participants, to perform genetic comparisons with the possible profiles found on the towels. DNA extracts were eluted out of the towels and genetic profiles were evaluated through the amplification of AmpFℓSTR®NGM SElect™Kit (Thermofisher™Scientific) kit markers.

Application of NGM detect™ PCR amplification kit for degraded forensic evidence samples

The Applied Biosystems™ NGM Detect™ PCR Amplification Kit is a new Human Identification solution with increased sensitivity in comparison to NGM SElect Kit.The purpose of this study is to evaluate the performance of the NGM Detect™ kit compared to AmpFℓSTR® NGM Select™ and AmpFlSTR® Identifiler® Plus kits, using a variety of casework samples. The study is being carried out on samples obtained from forensic cases such as sexual violence, exhumations, paternity, homicides and suspicious deaths. The sample typologies refer to: buccal swabs, nail swabs, swabs deriving from sexual violence, human biological tissues and liquids, clothes and objects of common use.The preliminary results show that the combined use of NGM Detect and NGM Select kits provide maximum information in a dual amplification strategy, as the size of several markers are complementary. Furthermore, the NGM Detect and NGM Select kits appear more sensitive than the Identifiler Plus kit. In conclusion, together with the NGM Select™ kit, the NGM Detect™ kit offers the possibility of recovering a lot of genetic information from degraded samples, delivering the best likelihood ratio value of identity compared to other ESS-focused solutions in the market today.

Optimization of the collection and analysis of touch DNA traces

The aim of this work was to optimize a strategy for detection, collection, and analysis of touch DNA traces. 4N6FLOWSwabs™ and cotton swabs were compared by collecting touch DNA traces from glass slides and gun shell casings. Shed cells were visualized using Diamond™ nucleic acid dye and a digital fluorescent microscope. Different collection and extraction methods were tested. Collected samples were amplified with the AmpFlSTR® NGMSElect™ kit and the Precision ID mtDNA Whole Genome Panel.

Analysis of 16 autosomal STR loci in Uyghur and Kazakh populations from Xinjiang, China

The AmpFℓSTR® NGM SElect™ Kit is a highly robust multiplex kit developed for human identification. This kit contains the expanded European Standard Set of Loci and the highly polymorphic SE33 locus. The high power of discrimination makes it one of the most informative kits for forensic applications. In order to evaluate the kit and get population data for Chinese Uyghurs and Kazakhs, the genetic polymorphisms of 16 autosomal STR loci included in the NGM SElect™ Kit: D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA, D10S1248, D22S1045, D2S441, D1S1656 D12S391, SE33, and Amelogenin were studied in 324 unrelated Uyghur and 124 unrelated Kazakh individuals from the Xinjiang Autonomous Region of China. Allele frequencies and forensic relevant parameters were calculated using the Arlequin software and DNAVIEW. The genotype frequency distribution of each locus did not deviate from Hardy-Weinberg Equilibrium in any of the two populations. The forensic parameters indicated that the kit is suitable for personal identification, paternity testing, and complex kinship analysis in Uyghur and Kazakh populations. Allele frequency data for the STR loci were compared with other previously published population data.

The Applied Biosystems™ NGM Detect™ PCR Amplification Kit – As promising as promised?

The new Applied Biosystems™ NGM Detect™ PCR Amplification kit comes with several innovations compared to the NGM SElect™ kit, e.g. a six color approach and rearranged loci, which makes it an interesting candidate for analysis of forensic case work samples. The internal validation shows that the shorter amplification time and the higher input volume are some of its main advantages, whereas the higher allelic imbalance is its main disadvantage.Following validation a concordance study with 1320 samples described in a publication by Dion et al. [3] was executed. The results with NGM Detect™ were compared to the ones with NGM SElect™ and PowerPlex® ESI 17 and revealed eight discordances between NGM Detect™ and NGM SElect™ and only one between NGM Detect™ and PP ESI 17. The concordance rate between the STR kits was greater than 99.982 %.

Automating PrepFiler® forensic DNA extraction kit: Optimization and validation on Freedom EVO® 150

To handle the growing number of forensic casework samples we decided to automate our trace sample workflow using PrepFiler® a magnetic bead based DNA extraction kit, on a Tecan liquid handling workstation Freedom EVO® 150. Even though there was a manufacturer-supplied validated protocol for the automated PrepFiler® DNA extraction, substantial changes were needed in order to obtain the optimal DNA yield and elution volume to meet the needs of our laboratory for further downstream applications. In this study, optimal lysis condition, elution volume and elution temperature were determined for the automated PrepFiler® extraction using blood samples on cotton swabs. Additionally, a comparison with the manual in-house method Chelex-100® was made using a variety of forensic mock samples on swabs. We tested blood, saliva and contact traces from a variety of substrates, with 10 replicates of each type. With the automated PrepFiler® system, the DNA yield obtained was around 30–70% lower than with Chelex-100®. However, the purity of the extracts was higher. Allele recovery of the STR profiles obtained with AmpFlSTR® NGM SElect™ kit was comparable. Furthermore we found that, besides the kit-specific optimization factors, swab characteristics are of major importance for the success of PrepFiler® DNA extraction. We incorporated the optimized PrepFiler® protocol on Freedom EVO® 150 into our routine workflow and included quality controls to maintain contamination-free and efficient DNA extraction with no evidence for loss of magnetic beads.

Forensic identification in teeth with caries

Human teeth are biological structures that resist extreme conditions thus becoming a useful source of DNA for human forensic identification purposes. When it is possible, forensic prefer only non-damaged teeth whereas those with cavities are usually rejected to avoid both external and internal bacterial contamination. Cavities are one of the most prevalent dental pathology and its incidence increases with ageing. The aim of this study was to validate the use of teeth with cavities for forensic identification. A total of 120 individual teeth from unrelated patients (60 healthy and 60 with cavities, respectively) extracted by a dentist as part of the normal process of treatment, were submitted for further analysis. Dental pulp was obtained after tooth fragmentation, complete DNA was extracted and the corresponding human identification profile was obtained by the AmpFlSTR® NGM SElect™ kit. Cariogenic microbiota was determined by PCR-DGGE with bacterial universal primers and bands were excised, re-amplified and sequenced. From the 120 dental pieces analyzed, a defined genetic profile was obtained in 81 (67.5%) of them, with no statistical differences between the healthy and the cavities-affected teeth. Statistical association between teeth status, DNA content and genetic profiles was not observed. Complex bacterial communities were only detected in the cavities group, being the Streptococcus/Enterococcus, and Lactobacillus genera the most represented. We conclude that teeth with cavities are as valid as healthy dental pieces for forensic human identification. Moreover, the severity of the cariogenic lesion as well as associated bacterial communities seems not to influence the establishment of human dental profiles.

Simultaneous Analysis of Micro‐RNA and DNA for Determining the Body Fluid Origin of DNA Profiles,

Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpFℓSTR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker.

Population genetic analyses of the STR loci of the AmpFlSTR NGM SElect™ kit for Han population in Fujian Province, China

Allele frequencies and forensically relevant population statistics of the STR loci in the AmpFlSTR® NGM SElect™ PCR Amplification Kit were estimated for the Han population from Fujian province in China (n = 454). All loci were highly polymorphic and the cumulative match probability was 5.4 × 10(-21). No significant departure from Hardy-Weinberg equilibrium and linkage equilibrium was detected after correction for sampling. The population substructure of Fujian Han population is minor.

Validation of the NGM SElect™ kit

The Swiss national database was launched August 2000 based on the 10 SGM Plus loci. With the aim of addressing the needs of the next-generation European STR genotyping systems in Switzerland, we validated the NGM SElect™ kit. In this study, we present the results of forensic validation studies including the following aspects: sensitivity, heterozygote peak height ratio calculations, performance with simulated PCR inhibition, proficiency tests and Swiss population data.

Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33

SE33 is one of the most informative markers in forensic use due to its high power of discrimination. During the course of developing the AmpFℓSTR® NGM SElect™ PCR Amplification Kit several SE33 primer designs were screened with one primer pair yielding a high frequency of discordant alleles when compared to the AmpFℓSTR® SEfiler Plus™ PCR Amplification Kit. This discordance was mostly specific to samples of African descent with an estimated frequency of 5.1% and was a result of a mobility shift of approximately +0.84nt. The sequence analysis of the affected alleles revealed that the only difference from the wild type sequence was a single nucleotide polymorphism (SNP) outside of the SE33 repeat but within the amplicon of this particular set of experimental primers. In total, we identified three different SNPs all within 9nt of each other, each of which could cause the mobility shift individually. Further characterization of this region via site directed mutagenesis and thermostability measurements strongly suggests that this polymorphic region contains a secondary structure that, when disrupted due to the presence of a variant SNP, results in a mobility shift relative to the wild type sequence. To overcome this problem, the SE33 primers used in the final configuration of the NGM SElect™ Kit avoided the amplification of this polymorphic region yielding in turn results highly concordant with the SEfiler Plus™ Kit.