Comparative study of five methods for DNA extraction from whole blood samples.
Abstract: Objectives

To find a suitable DNA isolation system from whole blood; five different DNA extraction methods were evaluated, comparing manual, partially automated and fully automated separation.

Methods

Phenol-chloroform, simple extraction by microwaving and the Wizard Genomic DNA Purification system were compared in this study. In addition to these manual methods, the MagNA Pure platform was performed manually, partially automated and fully automated. The last technique was a combination between the Wizard SV 96 Genomic DNA Purification Kit and the MagNa Pure extraction in which the whole vacuum system for waste collection was replaced by the magnetic separation principals.

Results

Best results were observed with the MagNa Pure automatic extraction that obtained nucleic acid amount of 0.98 [micro]g/[micro]l with 2.91 purity ratio. The second best methods were the magnetic beads based techniques, either the original or the modified one which showed purity ratios, 2.45 and 2.36 respectively and the amount of the nucleic acids recovered were 0.79 [micro]g/[micro]l and 0.76 [micro]g/[micro]l respectively.

Conclusions

Magnetic DNA separation is recommended for its high-quality nucleic acid extraction in which the risk of cross-contamination is reduced. It requires minimal starting material and is both cost-effective, user friendly and can be optionally automated with no manual intervention steps.

Keywords: DNA, isolation and purification, whole blood
Article Type: Report
Subject: Genetic research (Methods)
DNA testing (Laws, regulations and rules)
Blood (Medical examination)
Authors: Abd El-Aal, Amany A.
Abd Elghany, Nahed A.
Mohamadin, Ahmed M.
Badry, Aymen A. El-
Pub Date: 01/01/2010
Publication: Name: International Journal of Health Science Publisher: Renaissance Medical Publishing Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Renaissance Medical Publishing ISSN: 1791-4299
Issue: Date: Jan-March, 2010 Source Volume: 3 Source Issue: 1
Topic: Event Code: 930 Government regulation; 940 Government regulation (cont); 980 Legal issues & crime Advertising Code: 94 Legal/Government Regulation Computer Subject: Government regulation
Product: Product Code: 8000428 Blood Test Procedures NAICS Code: 6215 Medical and Diagnostic Laboratories
Accession Number: 225792680
Full Text: INTRODUCTION

Genomic DNA extraction is essential step prior to wide range molecular applications regardless of the molecular system chosen. DNA extraction method not only should yield DNA suitable for DNA quantity specifications but should provide the required amount of high-quality DNA with minimal contaminants which may interfere with the molecular technique. (1) The basic steps of DNA isolation are disruption of the cellular structure to create a lysate, separation of the soluble DNA from cell debris and other insoluble material and purification of this DNA from soluble proteins and other nucleic acids. Disruption of most cells is done by chaotropic salts, detergents or alkaline denaturation, and the resulting lysate is usually cleared by centrifugation, filtration or magnetic clearing. Historically, extraction of DNA was done using organic extraction (e.g. phenol-chloroform extraction, a liquid-liquid technique) followed by ethanol precipitation. (2) Other low cost laboratory methods were also used as thermal shock by boiling or microwaving. (3) On the other hand, a variety of commercially available kits are widely applied either manually, partially automated or by using a fully automated system.

In a trial to find a suitable DNA isolation system from whole blood, five different DNA extraction methods were evaluated in the present study ranging between manual, partially automated and fully automated separation.

MATERIALS AND METHODS

96 blood samples, 2 ml each, were collected within one week on EDTA tubes from patients attending the outpatient clinic in Taibah University, Saudi Arabia, for routine checking.

The DNA extraction methods used were as follows;

* phenol-chloroform purification method followed by an ethanol precipitation (2),

* simple extraction by microwaving based on thermal shock (3) and

* DNA extraction with the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA), which was carried out following the manufacturer's instructions.

* In addition to these three manual methods, the fully automated MagNA Pure platform based on magnetic glass (silica) particles was performed using LC Magna Pure Compact Instrument (Roche Diagnostics, Hoffmann-La Roche Ltd, USA).

* The previous technique was also applied manually and partially automated using the Precision[TM] XS Microplate Sample Processor from BioTek Instruments (Inc., P.O. Box 998, Highland Park, Winooski, Vermont 05404-0998 USA) for quick automatic washing.

* The last technique was a combination between the Wizard SV 96 Genomic DNA Purification Kit (Promega) and the MagNa Pure DNA purification methods, in which the whole vacuum system described by Promega for waste collection was replaced by the magnetic separation principals. The product was measured using spectrophotometer to be read on OD260 and OD280. Purity ratio was identified and nucleic acid [micro]g/[micro]l and [micro]g/ ml were measured. Moreover, time required, cost and simplicity were evaluated. To simplify determination of how much DNA was recovered, the input and output volume (100 [micro]l) for the DNA extraction was the same for each sample.

Brief description of the modified method

Whole blood sample was pipetted into each well of a 96 PCR microtiter plate (Promega) on an ambient 96 well pipetting position. "Combined Lysis Buffer" containing beads, proteinase K (Roche diagnostics) and Lysis Buffer were added to each sample and mixed thoroughly using the multichannel pipette. The plate was transferred to a heated position (55[degrees]C) for 15 minutes, using Dry Bath to aid cell lysis. The plate was then positioned on the separator magnet and left for 2 minutes to allow the beads to gather at the bottom of the tube. The supernatant was discarded by gentle pipetting from each well. The beads were washed (3- 4 times) by the addition of wash buffer while still on the magnet station to avoid disturbing the pellet. The wash buffer was removed by gentle pipetting of the contents of each well. Special care was taken to remove all residual liquid. The plates were allowed to air-dry for 5 minutes. Genomic DNA samples were eluted by resuspending the beads in Elution Buffer

Optional Step: Isolated genomic DNA can be separated from the beads by heating the PCR plate (65[degrees]C) for 10 minutes, and then transferring the clear DNA solution to a clean plate.

RESULTS

Tables 1 and 2 show the results of the different DNA extraction methods included in our comparative study. In general, there was a significant difference at the level of P <0.05 among different methods in each numerical parameter. Best results were observed with the MagNa Pure automatic extraction that obtained nucleic acid amount of 0.98 [micro]g/[micro]l with 2.91 purity ratio. The second best methods were the magnetic beads based techniques, either the original or the modified one which showed purity ratios, 2.45 and 2.36 respectively and the amount of the nucleic acids recovered were 0.79 [micro]g/[micro]l and 0.76 [micro]g/[micro]l respectively. The lowest purity was recorded with the quickest technique (microwaving) which showed also the lowest cost.

The time for MagNa Pure fully automated method was ranging from 60 to 90 minutes. The time of the manual magnetic separation and the modified method was about 2 hours in which repeated centrifugation or filtration were not needed.

DISCUSSION

Phenol-chloroform method and a microwave-based technique for DNA extraction were applied in the present study using the commercially available from laboratory supply houses materials with no need of extra supplies. Despite their low cost, the former is time consuming and used a variety of hazardous reagents (4) and the latter is an easy and rapid method but yield smaller DNA quantities compared to the other methods.

Orsini and Spica (5) concluded that microwave based method can be of value if applied to difficult substrates in environmental sources, including soil and sediments. In the present study the fully automated magnetic beads extraction obtained the best results compared to other methods with turnaround time for the whole process from 60 to 90 minutes, followed by the magnetic beads based methods either the original or the modified one with no need of centrifugation or filtration.

Nagy et al (6) recommended the use of magnetic beads extraction and reported that, linking the DNA to magnetic beads create an ensured specific interaction with the corresponding target and by means of an external magnet, the recovery of material for further studies is greatly simplified. On the other hand, one of the main problems which interfere with molecular reaction is inhibition; the efficiency of amplification of samples is often significantly decreased because of inhibitors might be present in the sample. Inhibition can result in partial DNA profiles, and in severe instances, false negative reactions. Many inhibitors are removed during the extraction process. Therefore, the choice of a suitable extraction method is crucial in molecular technology. (7)

Magnetic separation is considered one of the best methods recommended; in the present work, these methods obtained the best results concerning purity ratios, especially the fully automated technique. Pichl et al (4) reported that, magnetic beads method, demonstrate the ability to remove amplification inhibitors and use no hazardous chemicals. Moreover, Nagy et al (8) concluded that, full automation extraction can overcome many of the problems associated with the methods used so far. The previous authors added that, magnetic particles can also be used as adjuncts to traditional methods, when additional clean-up is warranted. In compliance with this concept, our modified technique in which the vacuum pumps or tubings were abolished from stage and were replaced by the magnetic separation policy. Therefore the risk of cross-contamination was reduced to an absolute minimum.

CONCLUSIONS

We conclude that optimization of DNA extraction protocol is essential to achieve best results and subsequent judgment regarding accurate molecular application. We recommend magnetic DNA separation for its high-quality nucleic acid extraction in which the risk of cross-contamination is reduced. It requires minimal starting material and is both cost-effective, user friendly and can be optionally automated with no manual intervention steps.

ACKNOWLEDGEMENT

Thanks to King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, for the great help and support throughout the whole study. (The research was approved and granted by the KACST grant no; AT 27-33).

Conflict of interest: None declared.

REFERENCES

(1.) Shutler GG, Gagnon P, Verret G, Kalyn H, Korkosh S, Johnston E, Halverson J. Removal of a PCR inhibitor and resolution of DNA STR types in mixed humancanine stains from a five year old case. J Forensic Sci. 1999 May;44(3):623-6.

(2.) Baechtel FS. The extraction, purification and quantification of DNA. In: Proceedings of the International Symposium on the Forensic Aspects of DNA Analysis. Washington, DC: U.S. Government Printing Office 1989, pp.25-28.

(3.) El-Awady MK, El-Hosseiny LA, Ismail M, Abdel-Azizand MA, El-Demellawy. elAwady MK, el-Hosseiny LA, Ismail SM, Abdel-Aziz MT, el-Demellawy MA. Comparison between Toxoplasma gondii DNA and specific immunoglobulins during pregnancy. East Mediterr Health J. 2000;6(5-6):888-97.

(4.) Pichl L, Heitmann A, Herzog P, Oster J, Smets H, Schottstedt V. Magnetic bead technology in viral RNA and DNA extraction from plasma minipools. Transfusion. 2005;45(7):1106-10.

(5.) Orsini M, Romano-Spica V.A microwave-based method for nucleic acid isolation from environmental samples. Lett Appl Microbiol. 2001;33(1):17-20.

(6.) Nagy M, Otremba P, KrugerC, Bergner-GreinerS, Anders P, Henske B, Prinz M, Roewer L. Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics.Forensic Sci Int. 2005;152(1):13-22.

(7.) Nagy B, Ban Z, Beke A, Nagy GR, Lazar L, Papp C, Toth-Pal E, Papp Z. Detection of Toxoplasma gondii from amniotic fluid, a comparison of four different molecular biological methods. Clin Chim Acta. 2006;368(1-2):131-7.

(8.) Al-Soud WA, Radstrom P.Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001;39(2):485-93.

Corresponding author: Dr. Amany Ahmed Abd El-Aal,

Professor of Parasitology,

Faculty of Medicine, Taibah University,

Kingdom of Saudi Arabia.

e-mail: dramanyahmed@hotmail.com

Amany A. Abd El-Aal [1], Nahed AAbd Elghany [2], Ahmed M. Mohamadin [2], Aymen A El- Badry [1] [1] Department of Parasitology & [2] Department of Biochemistry, Faculty of Medicine, Taibah University, Saudi Arabia
Table 1--Comparison between the different extraction methods as
regards the amount and purity of DNA yield

METHOD                 OD260   OD280   PURITY   NUCLIC     NUCLIC ACID
                                       RATIO    [mu]g/ml   [mu]g/ml
                                                ACID

Phenol-chloroform      0.99    0.72    2.22     727.40     0.72

Wizard Promega         0.91    0.69    1.78     697.90     0.69

Microwaving            0.82    0.50    1.31     508.50     0.50

Magna pure automatic   1.97    1.33    2.91     989.20     0.98

Magna Pure manual      1.34    0.86    2.45     791.0      0.79

Combined Promega &
MagNa Pure             1.29    0.81    2.36     761.0      0.76

Table 2--Comparative evaluation between the different DNA extraction
method

VARIABLES                       METHOD OF EXTRACTION

                Phenol-chloroform   Wizard            Microwaving
                                    Promega

Purity          2. 22               1.78              1.31

Quality of      727 [mu]g/ml        697               508
DNA/sample

Speed of        Many hours          About 2 hours     15 min
extraction

Simplicity of   Difficult,          Less simple       simple
extraction      need repeated
                centrifugations     centrifugations
                (6 times)           (3 times)

Cost            Medium              Moderate cost     v. low cost

VARIABLES                       METHOD OF EXTRACTION

                Magna               Magna             Combined Promega
                pure automatic      pure manual       & magna pure

Purity          2.91                2.45              2.36

Quality of      989                 791               761
DNA/sample

Speed of        60-90 min                      About 2 hour
extraction

Simplicity of   Extremely simple    More simple       More simple
extraction

Cost            The higher cost            Lower than automatic
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