Rescue PCR: Reagent-rich PCR recipe improves amplification of degraded DNA extracts

Johnson, Bobbi M.; Kemp, Brian M.

doi:10.1016/j.jasrep.2017.01.006

Cited by 9 publications

(16 citation statements)

References 56 publications

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“…This can be explained by DNA fragmentation and degradation due to treatment with alcohols and formaldehyde, which is carried out for fixation in paraffin blocks, and exposure to high temperatures when inadequate paraffin is used for the process. In addition, the high concentration of melanin present in the skin, and even more so in melanomas, is a possible inhibitor of PCR, since melanin can bind and inactivate at least a portion of the DNA to be amplified, limiting the binding of DNA polymerase and affecting both the amplification and speed of extension thereof 22,23 .…”

Section: Resultsmentioning

confidence: 99%

Melanoma: clinical-pathological and molecular analysis in patients of Ibague city, Colombia

Puentes

Estrada

Bohórquez

et al. 2020

Duazary

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This study aimed to establish the clinicopathological characteristics of patients with melanoma and its association with BRAF gene mutations. The pathology reports and paraffin-embedded tumor samples from 47 women and 30 men with melanoma, with an average age of diagnosis of 60 years, were reviewed at the Hospital Federico Lleras Acosta of Ibague, between 2010 and 2016. The presence of V600E mutation at the exon 15 of BRAF gene, was analyzed in these tumoral samples by Sanger sequencing and visual inspection of the electropherograms. We also studied the clinicopathological variables with X2, t-Student and the Kaplan Meier index. Most of the lesions were located in the lower limbs (46.6%). The most frequent subtype was Acral Lentiginous Melanoma (41.8%). Most lesions were of poor prognosis: Breslow depth greater than 4.1 mm (52.7%), ulceration (61.4%) and medium or high mitotic rate (> 30 %). The V600E mutation was identified in five patients with large, deep and ulcerated tumors, four of them had less than four years of survival. In conclusion, there was a higher frequency of melanoma in women, V600E BRAF mutation was present in patients with advanced disease (high Breslow index) and, the probability of five-year survival was less than 40%.

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Section: Resultsmentioning

confidence: 99%

Melanoma: clinical-pathological and molecular analysis in patients of Ibague city, Colombia

Puentes

Estrada

Bohórquez

et al. 2020

Duazary

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“…The insert length of silica gel dried material was limited by the size of DNA fragments by sonication (350 bp), whereas the insert length for herbarium material was shorter (250) probably due to degradation during drying and/or storage. There is potential for increasing the success of herbarium material by using an improved extraction protocol [48] or library preparation protocol [49,50], but this is not likely to improve the insert length. For nrDNA clusters, where the average cover was higher due to the higher number of copies in the cell, the assembly success was also higher.…”

Section: Effect Of Starting Materialsmentioning

confidence: 99%

The Treasure Vault Can be Opened: Large-Scale Genome Skimming Works Well Using Herbarium and Silica Gel Dried Material

Alsos

Lavergne

Merkel

et al. 2020

Plants

104

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Genome skimming has the potential for generating large data sets for DNA barcoding and wider biodiversity genomic studies, particularly via the assembly and annotation of full chloroplast (cpDNA) and nuclear ribosomal DNA (nrDNA) sequences. We compare the success of genome skims of 2051 herbarium specimens from Norway/Polar regions with 4604 freshly collected, silica gel dried specimens mainly from the European Alps and the Carpathians. Overall, we were able to assemble the full chloroplast genome for 67% of the samples and the full nrDNA cluster for 86%. Average insert length, cover and full cpDNA and rDNA assembly were considerably higher for silica gel dried than herbarium-preserved material. However, complete plastid genomes were still assembled for 54% of herbarium samples compared to 70% of silica dried samples. Moreover, there was comparable recovery of coding genes from both tissue sources (121 for silica gel dried and 118 for herbarium material) and only minor differences in assembly success of standard barcodes between silica dried (89% ITS2, 96% matK and rbcL) and herbarium material (87% ITS2, 98% matK and rbcL). The success rate was > 90% for all three markers in 1034 of 1036 genera in 160 families, and only Boraginaceae worked poorly, with 7 genera failing. Our study shows that large-scale genome skims are feasible and work well across most of the land plant families and genera we tested, independently of material type. It is therefore an efficient method for increasing the availability of plant biodiversity genomic data to support a multitude of downstream applications.

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“…Fig 8 shows that DNA extracted from heated WBC and BC minicapsules could be amplified by PCR with no indication of the presence of inhibitors since the PCR efficiency was constant and near 100% for both amplicons regardless the time points and temperatures [ 39 , 40 , 41 ] ( Table 3 ). Similar results were found for samples heated at 110°C.…”

Section: Resultsmentioning

confidence: 99%

“…A260 nm/ A230 nm ratios were also high for WBC and systematically lower for BC probably reflecting the fact that extractions are more challenging regarding contaminants when dealing with BC which were obviously contaminated by red blood cells [ 1 , 46 , 47 , 48 ][ 1 , 37 , 38 , 39 ].…”

Section: Discussionmentioning

confidence: 99%

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere

et al. 2017

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Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius’ law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats.

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Rescue PCR: Reagent-rich PCR recipe improves amplification of degraded DNA extracts

Cited by 9 publications

References 56 publications

Melanoma: clinical-pathological and molecular analysis in patients of Ibague city, Colombia

Melanoma: clinical-pathological and molecular analysis in patients of Ibague city, Colombia

The Treasure Vault Can be Opened: Large-Scale Genome Skimming Works Well Using Herbarium and Silica Gel Dried Material

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere

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