An efficient and safe method for the extraction of total DNA from shed frog skin

Xu, Yuan; Guan, Tianming; Liu, Jiayu; Su, Hang; Zhang, Zhen; Ning, Fangyong; Du, Zhiheng; Xiu-juan, Bai

doi:10.1007/s12686-019-01104-z

Cited by 7 publications

(7 citation statements)

References 10 publications

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“…DNA was extracted from the toes of individuals by phenol‐chloroform method described by Xu et al . ( 2019 ) for TRAP‐PCR.…”

Section: Methodsmentioning

confidence: 99%

Male heterogametic sex determination in Rana dybowskii based on sex‐linked molecular markers

Liu

et al. 2021

Integr. Zool.

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Identifying the mechanism for sex determination in amphibians is challenging. Very little is known about sex determination mechanisms of Rana dybowskii, a species of importance to evolutionary and conservation biology. We screened for sex‐linked molecular markers in R. dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1. We found 2 male‐linked molecular markers in R. dybowskii, which were 222 bp and 261 bp long. The detection rates of 222 bp marker in males form Xinglong, Huadian, and Dandong were 93.79%, 69.64%, and 13.64%, respectively, while the rate in females from Huadian was 27.50%. Besides, the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%, 87.50%, and 32.73%, respectively. The inheritance patterns of sex‐linked molecular markers showed that the 2 sex‐linked molecular markers were heterozygous. Compared to the XY‐male parent, progeny from XX‐pseudo‐male parent possessed lower sex reversal ratio at the same rearing temperature, and the proportion of female froglets from an XX‐pseudo‐male parent was more than 95% at low rearing temperature (15°C). Our findings suggest that R. dybowskii displays male heterogamety, and the 2 sex‐linked molecular markers may have a guiding significance for the protection and utilization of R. dybowskii.

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“…DNA was extracted from the toes of individuals by phenol‐chloroform method described by Xu et al . ( 2019 ) for TRAP‐PCR.…”

Section: Methodsmentioning

confidence: 99%

Male heterogametic sex determination in Rana dybowskii based on sex‐linked molecular markers

Liu

et al. 2021

Integr. Zool.

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“…A 'salting-out' protocol [62] was followed to extract genomic DNA for all pinned specimens of H. armigera. Though described more than 20 years ago, this method is still highly used and has recently been shown to produce higher DNA yields than other extraction methods, including phenol chloroform and kit-based [63]. I also found that the more expensive kit-based extraction methods gave equivalent starting concentrations to salting-out in a small test (data not shown).…”

Section: Genomic Dna Preparationmentioning

confidence: 94%

Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?

McGaughran

2020

BMC Genomics

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Background: Next generation sequencing (NGS) can recover DNA data from valuable extant and extinct museum specimens. However, archived or preserved DNA is difficult to sequence because of its fragmented, damaged nature, such that the most successful NGS methods for preserved specimens remain sub-optimal. Improving wetlab protocols and comprehensively determining the effects of sample age on NGS library quality are therefore of vital importance. Here, I examine the relationship between sample age and several indicators of library quality following targeted NGS sequencing of~1300 loci using 271 samples of pinned moth specimens (Helicoverpa armigera) ranging in age from 5 to 117 years.Results: I find that older samples have lower DNA concentrations following extraction and thus require a higher number of indexing PCR cycles during library preparation. When sequenced reads are aligned to a reference genome or to only the targeted region, older samples have a lower number of sequenced and mapped reads, lower mean coverage, and lower estimated library sizes, while the percentage of adapters in sequenced reads increases significantly as samples become older. Older samples also show the poorest capture success, with lower enrichment and a higher improved coverage anticipated from further sequencing. Conclusions: Sample age has significant, measurable impacts on the quality of NGS data following targeted enrichment. However, incorporating a uracil-removing enzyme into the blunt end-repair step during library preparation could help to repair DNA damage, and using a method that prevents adapter-dimer formation may result in improved data yields.

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“…DNA extraction was performed from web punches (4 mm in diameter except 6 months old for which we did 2 mm), according to Xu Y. et al [ 59 ] with minor modifications. Briefly, tissue chunks were digested o/n in a 1.5 ml tube containing 200 µl of lysis buffer (100 mM Tris-HCl pH 8; 200 mM NaCl; 0.20% SDS; 5 mM EDTA) + 4 µl of Proteinase K (20 mg/ml, NEB) at 55°C (300 rpm continuous shaking in a thermomixer).…”

Section: Methodsmentioning

confidence: 99%

Age-associated DNA methylation changes in Xenopus frogs

et al. 2023

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Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

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An efficient and safe method for the extraction of total DNA from shed frog skin

Cited by 7 publications

References 10 publications

Male heterogametic sex determination in Rana dybowskii based on sex‐linked molecular markers

Male heterogametic sex determination in Rana dybowskii based on sex‐linked molecular markers

Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?

Age-associated DNA methylation changes in Xenopus frogs

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