Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant ¨r)  system

Ewing, Margaret M.; Thompson, Jonelle M.; McLaren, Robert S.; Purpero, Vincent M.; Thomas, Kelli J.; Dobrowski, Patricia A.; DeGroot, Gretchen A.; Romsos, Erica L.; Storts, Douglas R.

doi:10.1016/j.fsigen.2016.04.007

Cited by 84 publications

(40 citation statements)

References 15 publications

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“…Further, none of the sperm fraction samples processed using the automated QIAcube method produced quantitation values where the total human DNA content was greater than 1.2 times more than the estimated male DNA content, whereas 72% and 30% of sperm fraction samples processed using the manual QIAGEN method and the manual organic method, respectively, produced values that indicated a substantial female contribution (>1.2:1 human:male ratios). This is important because evidence samples that result in human:male ratios greater than 1:1 are more likely to result in a mixed STR profile [32][33][34][35], which requires a more tedious and time-consuming interpretation process than that which is commonly needed for single source profile interpretation.…”

Section: Resultsmentioning

confidence: 99%

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

Goldstein

Cox

Seman

et al. 2019

Forensic Sciences Research

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Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6-8Â fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

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Section: Resultsmentioning

confidence: 99%

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

Goldstein

Cox

Seman

et al. 2019

Forensic Sciences Research

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“…However, the assay is based on high‐resolution melting and not on hydrolysis probes, which provide higher specificity. Commercial kits have shown sensitivity around 1 pg/µL [4–6]. That is achievable because they target multi‐copy genomic loci and the sequence of the oligonucleotides is unknown to the public.…”

Section: Discussionmentioning

confidence: 99%

“…For over 10 years, multiplex qPCR for DNA quantitation has been reported in forensics [2] and it is considered the gold standard for human‐specific DNA quantitation. Nevertheless, the assays have been developed and validated on platforms that detect more than three different dyes [3–9]. With the spreading of qPCR technology, smaller, more economic and user‐friendly platforms have reached the market.…”

Section: Introductionmentioning

confidence: 99%

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

et al. 2020

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DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratoriesFor over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

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“…Kadar hkrati pomnožujemo kratek in dolg fragment DNA, lahko iz njunega razmerja izraču-namo tudi stopnjo razgrajenosti DNA, ki smo jo pridobili iz posmrtnih ostankov, kar nam pomaga pri izbiri genetskih označevalcev, ki jih bomo tipizirali. Poleg količine DNA v vzorcu in stopnje njene degradiranosti, lahko z novejšimi kvantifikacijskimi kompleti ugotavljamo tudi prisotnost inhibitorjev reakcije PCR v ekstraktu (29). Če je začetna koncentracija DNA v vzorcu zelo nizka, se zaradi stohastičnega učinka možnost napak pri pomnoževanju v reakciji PCR poveča, saj lahko pride do izpada alelov zaradi nepomnožitve, kar moramo upoštevati pri interpretaciji rezultatov.…”

Section: Določitev Količine Dna V Vzorcihunclassified

Genetska identifikacija pogrešanih oseb

Bajželj

Pajnič

2017

SlovMedJour

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IzvlečekPrispevek na pregleden način opisuje identifikacijo pogrešanih oseb iz neprepoznavnih posmrtnih ostankov, ki jo opravimo z molekularno-genetskimi metodami. Izredno polimorfna, individualno specifična področja, ki nam omogočajo prepoznavo pogrešanega, so mikrosateliti na avtosomih in kromosomu Y ter kontrolna regija mitohondrijske DNA. Za primerjavo genetskih profilov je potrebno odvzeti biološki material iz posmrtnih ostankov in pridobiti primerjalne referenčne vzorce. Če so ostanke našli kmalu po domnevni smrti pogrešanega, poskušamo pridobiti primerjalne vzorce z njegovih osebnih predmetov. Če teh ni, uporabimo kot referenčne vzorce brise ustnih sluznic njegovih sorodnikov ali morebitna, v zdravstvenih ustanovah arhivirana tkiva, če je bila v času življe-nja pogrešane osebe opravljena biopsija za potrebe medicinske diagnostike. Kadar ni ne osebnih predmetov, ne svojcev in ne arhiviranih tkiv pogrešane osebe, genetska identifikacija ni možna. Kot biološki material najpogosteje uporabimo kri, mehka tkiva, nohte, zobe ali kosti, kar je odvisno od stanja posmrtnih ostankov. Postopki, ki jih uporabljamo za ekstrakcijo DNA iz različnih tkiv, se med seboj razlikujejo po zahtevnosti in času, ki ga porabimo za pridobitev DNA. Najzahtevnejša in časovno potratna je izolacija DNA iz kosti in zob, zato se zanjo odločimo le v primeru skeletiziranih posmrtnih ostankov, ali ko iz drugih tkiv ne uspemo pridobiti zadostne količine DNA za genetsko identifikacijo. Ob ujemanju genetskih profilov neprepoznavnih posmrtnih ostankov in primerjalnih vzorcev pogrešane osebe je potrebno moč genetskega dokaza statistično ovrednotiti in podati verjetnost identifikacije. AbstractThis article presents identification of missing persons from badly preserved post-mortem remains using molecular genetics methods. Extremely polymorphic and individually specific genetic markers that enable the identification of missing persons are microsatellites on autosomal chromosomes, microsatellites on Y chromosome and control region of mitochondrial DNA. For genetic profile comparison, biological material from post-mortem remains and reference samples have to be collected. If post-mortem remains are found shortly after the presumed death of the missing person, their personal items are used for comparison. If these are not available, (the missing person's) relatives could be used as reference samples or achieved tissues stored in medical institutions if biopsy for the needs of medical diagnostics was performed earlier during their life. When reference samples are not available, genetic identification is not possible. The type of biological material sampled from the deceased depends on the condition of human remains. Blood, soft tissues, nails, teeth or bones are most commonly used for genetic identification, and the time required for DNA extraction depends on the type of biological material. The most demanding and time consuming is extraction of DNA from teeth and bones, therefore we use it in cases when only skeleton is available or we cannot get a su...

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Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant ¨r) system

Cited by 84 publications

References 15 publications

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

Genetska identifikacija pogrešanih oseb

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