Human DNA degradation assessment and male DNA detection by quantitative-PCR followed by high-resolution melting analysis

Ginart, Santiago; Caputo, Mariela; Corach, Daniel; Sala, A.

doi:10.1016/j.forsciint.2018.11.013

Cited by 13 publications

(11 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1a shows melting profiles of DNAs controls, males and female donors and how Q1a3a NA-Y-hg is discriminated. Long and short autosomal fragments melting peaks (Tm≈ 83 °C; Tm≈ 84.8 °C, respectively) are useful as quality indicators of DNA integrity [2]. The ability to generate quality genetic profiles was demonstrated by STRs results obtained after DNA markers amplification where DNA template was normalized according to the quantification results of this tool (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…25 μL final volume: 37.5 pmol Syto9(Invitrogen, USA), 1.25 units GoTaq DNAPolymerase, 5X Colorless GoTaq Reaction Buffer, 3.75 μmol 4dNTPs mix (Promega), 1.7 mM Mg 2+ and 2 μL sample DNA. Primers: 8.5 pmol Y-chromosome amplicon [6], 10 pmol short autosomal amplicon [2] ; 12.5 pmol long autosomal amplicon [2]. qPCR platform: Mic qPCR Cycler (Bio Molecular Systems Ⓒ Australia).…”

Section: Reagents and Amplification Conditionsmentioning

confidence: 99%

“…Previous developments of quantification systems allowed us to obtain an efficient tool to simultaneously quantify human DNA, detect male DNA and estimate DNA degradation in one real time PCR reaction followed by high resolution melt (HRM) using Syto9 chemistry [1][2][3]. Farther optimization of this approach allowed us to obtain additional information for screening purposes.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Q1a3a native-American Y-haplogroup detection in DNA quantification step: A quick diagnosis for Y-chromosome database selection

Ginart

Caputo

Corach

et al. 2019

Forensic Science International: Genetics Supplement Series

Self Cite

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

Section: Reagents and Amplification Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Q1a3a native-American Y-haplogroup detection in DNA quantification step: A quick diagnosis for Y-chromosome database selection

Ginart

Caputo

Corach

et al. 2019

Forensic Science International: Genetics Supplement Series

Self Cite

View full text Add to dashboard Cite

“…The studied qPCR assay sensitivity is the same as the original publication [3] and remained when half‐volume reactions were performed (unpublished data). A recently published qPCR assay for DNA quantitation and degradation assessment [16,17] reported a sensitivity of 3.2 pg/µL. However, the assay is based on high‐resolution melting and not on hydrolysis probes, which provide higher specificity.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

View full text Add to dashboard Cite

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.

show abstract

“…DNA was extracted by three methods: a-decalcification [6] (n = 3); b-total bone demineralization [7] (n = 3) and c-purification by a semi-automated process [8] (n = 3). Quantification was performed by fluorimetry (Quantus™ Fluorometer, Promega, Madison, USA) (F), real time PCR (Plexor® HY, Promega, Madison, USA) (P) and with an "in-house" real time PCR method (H) that allows quantification DNA concentration, detects Y-chromosome markers and assess degradation ratio [9]. Amplification was performed with Global Filer and detected in ABI 3500.…”

Section: Methodsmentioning

confidence: 99%

Salty or sweety? Alternatives for bone preservation along extended periods of time

Caputo

Garrigós

Ginart

et al. 2019

Forensic Science International: Genetics Supplement Series

View full text Add to dashboard Cite

Sodium chloride is an effective human corpse tissue preservation medium due to its hygroscopic features and other phyco-chemical characteristics. We present herein sucrose as an alternative material for this purpose. Both media: solid Sodium Chloride and sucrose were tested comparing their efficiency as preservation alternatives. We used a set of 18 phalanges of a female hand buried for over ten year in a sealed coffin. After exhumation, 9 phalanges were preserved in sugar and 9 in salt, individually stored in 15 ml polypropylene tubes at room temperature for 4 years. DNA was extracted by three methods: a-total bone decalcification; b-total demineralization and c-purification by a semi-automated process. Quantification was performed by fluorimetry (F), Plexor (P) and with an "in house" method (H). Amplification was performed with Global Filer and detected in an ABI 3500. DNA yield by method a-(F: 1.36 ± 0.93 ng/ul; P:0.23 ± 0.18 ng/ul) was higher than for b-(F:0,99 ± 0.31 ng/ul; P: 0,14 ± 0.13 ng/ul) or c-method (not detected). Amplification efficiency was determined by the percentage of complete markers (22 loci), the inter-loci balance _ILB-(Shannon Entropy; SH: 3.09 as maximum) and intra-locus balance (mean local balance: MLB). The inter-loci balance was higher for preservation in sugar than in salt (SH: 1.69 vs 1.07) while MLB values did not show significant differences (0.88 vs. 0.89). Degradation ratio correlates with the genetic profile quality. Sugar, as an alternative conservative medium combined with DNA extraction by method a-showed an optimal strategy to obtain good quality genetic profiles.

show abstract

Human DNA degradation assessment and male DNA detection by quantitative-PCR followed by high-resolution melting analysis

Cited by 13 publications

References 14 publications

Q1a3a native-American Y-haplogroup detection in DNA quantification step: A quick diagnosis for Y-chromosome database selection

Q1a3a native-American Y-haplogroup detection in DNA quantification step: A quick diagnosis for Y-chromosome database selection

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

Salty or sweety? Alternatives for bone preservation along extended periods of time

Contact Info

Product

Resources

About