Referee report. For: Optimization of extraction of genomic DNA from archived dried blood spot (DBS): potential application in epidemiological research & bio banking [version 1; referees: 1 not approved] (original) (raw)
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Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots
Journal of Clinical Laboratory Analysis, 2016
Background: Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysissalting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. Methods: For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from these DBS samples by SLSO, QIAamp R gDNA Micro kit and in-nuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. Results: The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. Conclusion: The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies.
DNA Extraction and Stability for Epidemiological Studies
Clinical Chemistry and Laboratory Medicine, 1998
During the last few years, an important development of molecular biology techniques has been observed in research and clinical laboratories, and consequently the availability of DNA is becoming essential for epidemiological studies. Several DNA extraction procedures have been proposed. However as the quantity and quality of the DNA extracted is very variable, standardization of the storage of samples, and of extraction procedures becomes essential. Three steps will be considered. (i) Procedures of whole blood preservation prior to extraction which seem to affect yield and purity of the DNA extracted; (ii) DNA extraction procedures, which must be validated by a systematic DNA quality control using DNA molecular weight screening and spectrophotometric analysis, and also by verification that the DNA obtained is well adapted for molecular biology applications; (iii) the third important factor to study is the storage of DNA, since data on short-term (several months) and especially on lon...
Extracting DNA from long term stored (LTS) whole blood samples remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. In this study, we compared three methods of gDNA extraction methods, from which mechanical (glass bead) method is suitable for gDNA extraction from LTS whole blood sample of coronary artery disease patient. Mechanical (glass bead) method is cost effective with less time consuming method and also gives good yield of gDNA.
A comparative evaluation of four DNA extraction protocols from whole blood sample
Cellular and molecular biology, 2016
All organisms have Deoxyribonucleic acid (DNA) within their cells. DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. DNA extraction is one of the most basic and essential techniques in the study of DNA that allow huge advances in molecular biology, biotechnology and bioinformatics laboratories. Whole blood samples are one of the main sources used to obtain DNA and there are many different protocols available in this issue. In current research, compared four DNA extraction protocols from blood samples; include modified phenol-chloroform protocol, two salting-out and enzyme free method and from commercial kit. The extracted DNAs by these protocols were analyzed according to their time demands, quality and quantity, toxicity and functionality in PCR method. Also the quality and quantity of the extracted DNA were surveyed by gel electrophoresis and Nanodrop spectrophotometry methods. It was observed that there are not significant...
PLOS ONE, 2015
Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.
Molecular and Cellular Probes, 2011
Long-term stored (LTS) whole blood collection can be an important source of DNA without collection costs, but there is a lack of information on methods useful to extract genomic DNA from such type of biological material. Here we report a simple and fast revisited phenol/chloroform extraction method from LTS whole blood. Protocol reliability was assessed by comparison with proteinase K and silicagel membrane spin column-based DNA extraction methods using LTS À20 C whole blood from 1980, and by testing it on 82 whole blood samples, collected from 1980 to 1995, with high quality (A 260/280 ¼ 1.79 AE 0.32 O.D., A 260/230 ¼ 1.45 AE 0.52 O.D.) and quantity results. Genotyping efficiency was also checked by performing RFLP-PCR and ASP-PCR of p53 Pro72Arg (rs1042522) SNP and hTERT MNS16A VNTR, respectively, resulting in 100% of samples successfully typed. In addition to the goodness and the efficiency of method proposed here, this protocol achieves working time reduction combining extraction and purification steps, allowing to work at room temperature. Furthermore, phenol is able to inactivate any potential nuclease and potential infective sources from the first step on. Based on these results we also conclude that LTS À20 C whole blood samples may be considered a reliable and potential resource for future genotyping studies and retrospective analysis in a genetic epidemiological setting.
An Efficiency Human Genomic DNA Extraction from Dried Blood Spots
Procedia Environmental Sciences, 2011
Extraction of genomic DNA is the very first experiment in the study cascade of human inherited diseases. Blood is preferred to be the material for DNA extraction. In the case of limitation of the material source, for example, new born screening program, blood is saved as dried spot in filter paper. In practical, long term storing blood sample as the dried spots on filter paper leads to more advantages for DNA extraction essay which need to be performed several times. This study provides an efficient method with 2 step lyses allow high concentration of DNA is extracted from samples. Comparing to the commercial QIAamp® DNA Mini Kit DNA concentration extracted from this method is higher, the quality of DNA is equal and the cost is lower. The optimal DNA extraction method based on phenol in this study is useful tool which may apply for further molecular analysis studies using dried blood spot samples.
Comparison of manual and automated nucleic acid extraction from whole-blood samples
Journal of Clinical Laboratory Analysis, 2007
Nucleic acid extraction and purification from whole blood is a routine application in many laboratories. Automation of this procedure promises standardized sample treatment, a low error rate, and avoidance of contamination. The performance of the BioRobot M48 (Qiagen) and the manual QIAmp s DNA Blood Mini Kit (Qiagen) was compared for the extraction of DNA from whole blood. The concentration and purity of the extracted DNAs were determined by spectrophotometry. Analytical sensitivity was assessed by common PCR and genotyping techniques. The quantity and quality of the generated DNAs were slightly higher using the manual extraction method. The results of downstream applications were comparable to each other. Amplification of high-molecular-weight PCR fragments, genotyping by restriction digest, and pyrosequencing were successful for all samples. No cross-contamination could be detected. While automated DNA extraction requires significantly less hands-on time, it is slightly more expensive than the manual extraction method.
Comparison of DNA extraction methods for samples from old blood collections
2021
In this study, DNA was extracted from whole blood which had been collected and stored at -20°C for 5-18 years, with the aim of determining the most suitable commercial DNA extraction kit for this purpose. DNA from nine cord blood samples collected in 1999, 2001 and 2012, with low blood volumes (<1 ml), and a partly dried adult blood sample collected in 2003, having a large blood volume (6 ml) was extracted using four different DNA extraction kits: Quick-DNA Miniprep Plus kit, DNeasy Blood & Tissue kit, MagAttract HMW DNA kit and QIAamp Blood Maxi kit. We concluded that high-quality DNA can be extracted from whole blood sample collections which have been stored for even up to 18 years in a biobank at -20°C.
A method for improving the efficiency of DNA extraction from clotted blood samples
Journal of Clinical Laboratory Analysis, 2019
BackgroundThe efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods.MethodsInitially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the conce...