Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Meredith, Melanie; Bright, Jo‐Anne; Cockerton, Sarah L; Vintiner, Sue

doi:10.1016/j.fsigen.2011.02.007

Cited by 28 publications

(14 citation statements)

References 10 publications

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“…A DNA fragment will only be visualized if there are sufficient molecules present to trigger the capillary electrophoresis machine’s CCD camera. For 28 cycles, approximately 30 haploid copies (ca 90 pg) are required before sufficient PCR product is available to trigger a signal [43], whereas for 34 cycles, just one molecule (ca 3 pg in a haploid cell) is needed to produce sufficient signal [44]. …”

Section: Resultsmentioning

confidence: 99%

Characterization of degradation and heterozygote balance by simulation of the forensic DNA analysis process

2016

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Simulation experiments were used to show the impact of varying extraction efficiency, aliquot proportion, and PCR efficiency on the heterozygote balance of a range of diploid and haploid cells. Reducing either parameters introduces variance. It is well-known that the variance in heterozygote balance increases as the amount of DNA is reduced. Surprisingly the distribution is in fact diamond shaped — the variance start to decrease at very low amounts of DNA. Simulations suggest that pristine diluted DNA is an acceptable approximation in validations to infer heterozygote balance. However, the difference in distribution of the variance between diploid and haploid cell types may, under some circumstances, need to be considered in statistical models. Finally, we exemplify how simulations can be used to predict the outcome of PCR for degraded samples. Visualizing the predicted DNA profile as an electropherogram can help to identify the best approach for sample processing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-016-1453-x) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Characterization of degradation and heterozygote balance by simulation of the forensic DNA analysis process

2016

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“…The procedures are presented below:

Direct lysis method: the cells were suspended in 15 μl PBS and incubated at 95°C for 15 min; the supernatant was used for PCR directly after centrifugation at 12,000 rpm for 1 min.

PK method: this method was performed as described before with modifications [12,14]. Briefly, the cells were suspended in 15 μl lysis buffer (10 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 0.2M NaCl and 1 mg/ml proteinase K), followed by 55°C for 60 min and then 95°C for 15 min; the supernatant was used for PCR directly after centrifugation at 12,000 rpm for 1 min.

PK-T20 (Proteinase K-Tween 20) method: this method was performed as described before with modifications [11]. Briefly, the cells were suspended in 15 μl lysis buffer (10 mM Tris-HCl pH8.0, 2 mM EDTA pH 8.0, 2% Tween 20, and 1 mg/ml proteinase K), followed by 55°C for 60 min and then 95°C for 15 min; the supernatant was used for PCR directly after centrifugation at 12,000 rpm for 1 min.…”

Section: Methodsmentioning

confidence: 99%

“…PK-T20 (Proteinase K-Tween 20) method: this method was performed as described before with modifications [11]. Briefly, the cells were suspended in 15 μl lysis buffer (10 mM Tris-HCl pH8.0, 2 mM EDTA pH 8.0, 2% Tween 20, and 1 mg/ml proteinase K), followed by 55°C for 60 min and then 95°C for 15 min; the supernatant was used for PCR directly after centrifugation at 12,000 rpm for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Rapid Identification of Genome-Edited Mesenchymal Stem Cell Colonies via Cas9

et al. 2019

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Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previously optimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSC colonies over-expressing IL-10 in the AAVS1 locus.

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“…[1][2][3][4], many of them using vaginal or even mouth buccal swabs spiked with known amounts of semen. Whilst it might be more convenient to procure samples of such swabs and load these with semen, rather than collect true PC swabs, we have demonstrated that such mock samples do not behave in the same way as a true PC sample.…”

Section: Post-coital Samplesmentioning

confidence: 99%