* E-mail: androniki.psifidi@roslin.ed.ac.uk Affiliations Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom ⨯
Affiliation Microbiology and Infectious Diseases Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece ⨯
Affiliation Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece ⨯
Affiliation Food safety Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece ⨯
Affiliation Department of Clinical Veterinary Sciences, University of Bristol, Langford House, Langford, Bristol, United Kingdom ⨯
Affiliation Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece ⨯
Affiliations Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom, Scotland’s Rural College, Edinburgh, United Kingdom ⨯
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.
Citation: Psifidi A, Dovas CI, Bramis G, Lazou T, Russel CL, Arsenos G, et al. (2015) Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples. PLoS ONE 10(1): e0115960. https://doi.org/10.1371/journal.pone.0115960
Received: September 25, 2014; Accepted: November 28, 2014; Published: January 30, 2015
Copyright: © 2015 Psifidi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: The data is all contained within the paper.
Funding: This research was funded by the Seven Framework Program of the European Commission, project 3SR (Sustainable Solutions for Small ruminants). 3SR Grant agreement no.: FP7-KBBE-2009-3-245140. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The successful completion of the Human Genome Project and the achievement of similar goals in other species have generated a huge amount of freely available information about the genomic sequence of different organisms, opening the door to a post-genomic era where new challenges arise [1,2]. This new era is also characterized by the development of new technologies which enable the study of thousands of genes and/or molecular markers at once. Such a technology is based on DNA microarrays, which is a multiplex technique used for rapid, large-scale genotyping. This technique has fast become a standard approach in molecular biology research and clinical diagnostics [3]. Microarrays have already been successfully applied in as diverse scientific studies as cell biology, molecular microbiology, cancer genetics, genetic and metabolic disorders, infectious diseases, drug discovery, host-pathogen interaction, population genetics, linkage analysis, genetic improvement of livestock species, evolutionary biology, detection of food-borne pathogens, stress responses, forensic analysis and toxicological research [3–9].
In the last few years, further enormous advances in genotyping technology have been taking place with the development of the next generation sequencing (NGS) technologies. Whole genome sequencing provides information on a genome that is orders of magnitude larger than that provided by DNA microarrays [10]. To date, these technologies have been applied in a variety of contexts, including whole genome sequencing, de novo genome sequencing, exome sequencing, targeted resequencing, cancer cell sequencing, de novo transcriptome sequencing, RNA sequencing, small RNA sequencing, metagenomic sequencing and microbial strain screening, among others [11–18] (http://www.beckmangenomics.com/genomic_services/next_generation_sequencing/). Although NGS platforms are improving at a very quick rate, thereby reducing costs by a factor of two to three each year, the cost is still too high for routine large-scale sequencing of whole genomes for scientific research [19]. At this point, next generation platforms are usually used as complementary to microarray analysis.
Microarray technology has been improved significantly in that period, in terms of diminished cost and sample requirement, and has yielded increased data density and quality [20]. However, it still remains a complex process that is prone to technical difficulties if reagents and input material are not of suitable quality [21,22]. The first crucial step for microarray analysis is considered to be DNA extraction and quality control of the extracted nucleic acids. Whole-genome microarray analysis continues to require an input DNA mass that is at least 100 times larger than that required for simple PCR testing and requires very pure DNA that is double stranded with a length span at least 5 times longer than required for most PCR reactions [23]. Usually, a DNA quantity of 2.5 to 3.0 μg is necessary depending on the array size and platform used (http://www.ark-genomics.org/news/edinburgh-genomics). However, when other panels and techniques are used for whole genome genotyping, like KASP genotyping, a higher quantity of DNA, up to 6.0 μg, is required (http://www.lgcgenomics.com/genotyping/kasp-genotyping-chemistry/genotyping-panels). Similarly, in the case of NGS, DNA quantity requirements differ depending on the genotyping aim and the platform used. For whole genome de novo sequencing, which is used to sequence uncharacterized genomes where there is no reference sequence available or known genomes where significant structural variation is expected like in cancer cells, a very high DNA quantity is required, usually from 30 to 60 μg depending on the platform. For whole genome sequencing, usually a quantity above 10 μg, ideally 20 μg DNA is desirable, while for targeted resequencing of custom regions of interest a lower DNA quantity of about 3 to 6 μg is used [12] (http://genepool.bio.ed.ac.uk/illumina/samples.html). A minimum concentration of 50 ng/μl is also necessary in both microarray and NGS analysis. Picogreen assay with Qubit platform is considered to be the method of choice for DNA quantification. Implementation of quantification methods other than Picogreen may lead genotyping companies to ask for more concentrated and higher amounts of DNA for the analysis (http://genepool.bio.ed.ac.uk/illumina/samples.html). A gel photo documenting high quality DNA is required to accompany the samples, as well. Although the different NGS and microarray platforms have specific requirements regarding DNA quantity, purity and integrity in order to achieve reliable genotyping data, no specific guidance on the protocol of choice is given by the genotyping centers. The need for robust methods that produce a representative, non-biased source of nucleic acid material from the genome under investigation is acknowledged [12].
Another important aspect regarding DNA extraction protocols and advanced genotyping analysis is the suitability of the extracts for long term DNA-banking. Usually DNA extracts have to be stored until all samples are collected, which differs among the studies, and until the genotyping centers have capacity available. Moreover, there is an increased interest in the creation of DNA banks since sample collection and DNA extraction are laborious, expensive and time consuming procedures. Storage tests carried out by the DNA Bank Network revealed that high purity of extracted DNA must be ensured, since secondary compounds and heavy metal ions can result in highly reactive intermediates causing all sorts of DNA damage [24,25].
Although, the selection of an appropriate DNA extraction method plays a pivotal role in the success of genome-wide studies and long term DNA-banking, there are no established standard operating procedures for genomic DNA extraction. Moreover, there are no published reports on simultaneous comparisons of the efficiency of different genomic DNA extraction procedures for microarray analysis or NGS applications, and only a few studies in the literature that compare different extraction protocols for microbial DNA suitable for microarrays analysis [21,26–29].
The objective of this work was to evaluate eleven different methods for extraction of genomic DNA from ovine blood samples in terms of DNA quantity, concentration, purity, integrity and real-time PCR suitability, as well as utility and applicability for subsequent DNA microarray genotyping and long–term storage.
At first, 11 blood samples were taken from each of 16 ewes of the Chios dairy breed raised in an experimental flock. These samples were used to evaluate the DNA extraction methods described next. Peripheral blood samples were collected in 9 ml K2EDTA Vacutainer blood collection tubes (BD diagnostics) by jugular venepuncture. These samples were inverted to mix and prevent clotting and immediately placed in isothermic boxes and transferred to the laboratory. Individual blood samples from the same animal where mixed together and then divided again in order for each blood sample to contain the same amount of leucocytes. At the end of the procedure, all 16 animals had DNA extracted with each one of 11 DNA extraction methods described in detail in the next section.
Three of the DNA extraction methods (Nucleospin Blood, Nucleospin Blood L, Nucleospin Blood XL, Macherey-Nagel, Duren, Germany) used whole blood as source of genomic DNA while the rest of them used buffy coat (Table 1). In the latter cases, buffy coat was prepared by spinning whole blood at 3,000 g for 10 min in an Eppendorf (5415R) centrifuge (Hamburg, Germany) at room temperature to separate the blood into its plasma, leukocyte and erythrocyte fractions. The buffy coat was removed and dispersed in 700 μl of red cell lysis buffer (25 mM NaHCO3, 0.3 M NH4Cl, 5 mM EDTA). A second centrifugation at 3,000 g for 10 min at room temperature followed. The liquid was discarded and the leucocyte pellet was dispersed in different buffers depending on the DNA extraction method followed.