Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques

Panda, Barsa Baisalini; Pradhan, Nitika; Hazra, Rupenangshu K.

doi:10.1007/s11033-018-4456-5

Cited by 8 publications

(4 citation statements)

References 21 publications

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“…This suggests that DNA fragmentation, similar to DNA strandedness, is also affected by factors such as storage and original sample conditions, in addition to sample type and applied DNA extraction protocol. While numerous studies have shown that Chelex-based extraction protocols are cost-effective with similar or better performance compared to commercially available kits [6, 52, 73-78], concerns have been raised whether Chelex-based extracts provide adequate DNA stability for long-term storage [79]. The high pH (10-11) of the Chelex solution has been shown to decrease over time [80], and the chelating effect of the Chelex resin, removing metal cations required for DNase activity, may also decline over time.…”

Section: Resultsmentioning

confidence: 99%

Assessment of DNA quality for whole genome library preparation

Jansson,

Fagerholm,

Börkén

et al. 2024

Preprint

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In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

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Section: Resultsmentioning

confidence: 99%

Assessment of DNA quality for whole genome library preparation

Jansson,

Fagerholm,

Börkén

et al. 2024

Preprint

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“…It chelates metal ions that are crucial for the activity of nucleases . Chelex‐100 has been widely used for simplified DNA extraction from a great number of biological and non‐biological samples such as carbonized and decomposing corpse, mosquito abdomen, cigarette butts, semen, bloodstain and buccal swab, which produce varying DNA yield and quality. In our case, the use of Chelex‐100 neither in water nor in Tris‐buffer did not contribute to the efficiency of cell lysis.…”

Section: Resultsmentioning

confidence: 99%

Potential authentication of various meat‐based products using simple and efficient DNA extraction method

et al. 2020

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BACKGROUND: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR.RESULTS: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R 2 = 0.9979) based on the regression analysis of the standard curve have been obtained.CONCLUSION: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market.

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“…In addition, extraction of samples with an organic solvent such as chloroform and their precipitation with ethanol allow to obtain DNA that can be used for SNP-RFLP genotyping studies. Previous studies developed similar extraction methods for detection and genotyping of parasites [12,13], but the advantage of our method is the ability to obtain signi cant quantities of DNA, in fact mean DNA yields are 2-3 µg, a result until now obtained with commercial kits [14,15]. In 2018, Panda et al compared three methods for DNA extraction from mosquitoes dried blood spots: Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer, obtaining the lowest yield with TE buffer method [13].…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies developed similar extraction methods for detection and genotyping of parasites [12,13], but the advantage of our method is the ability to obtain signi cant quantities of DNA, in fact mean DNA yields are 2-3 µg, a result until now obtained with commercial kits [14,15]. In 2018, Panda et al compared three methods for DNA extraction from mosquitoes dried blood spots: Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer, obtaining the lowest yield with TE buffer method [13]. The TE buffer-based DNA extraction method described here has instead shown better results, compared with traditional protocols.…”

Section: Discussionmentioning

confidence: 99%

A Protocol for Rapid and Inexpensive Genotyping of Mutant Mice from Dried Blood Spots: An Update

Romano

Zuchegna

Zannini

et al. 2021

Preprint

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Background: Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a homemade DBS method followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). Results: We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the sensitivity, specificity and feasibility of this novel procedure. Conclusions: Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a miniscule amount of sample.

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Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques

Cited by 8 publications

References 21 publications

Assessment of DNA quality for whole genome library preparation

Assessment of DNA quality for whole genome library preparation

Potential authentication of various meat‐based products using simple and efficient DNA extraction method

A Protocol for Rapid and Inexpensive Genotyping of Mutant Mice from Dried Blood Spots: An Update

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