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Rapid Extraction of High Quality DNA from Whole Blood Stored at 4C for Long Period

Author: V. Iranpur M.1,*, A. K. Esmailizadeh
Affiliation: 1 Department of Animal Science, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran
2 Department of Animal Science, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.
3 Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran.
*Author for correspondence: Vahid Iranpur-mobarakeh, Department of Animal Science, Faculty of Agriculture, Shahrekord University, Shahrekord, P.O.Box 115, Iran, e-mail: iranpur2010@yahoo.com Fax number: +98 3814424412
 

Source: Vahid Iranpur-mobarakeh, Department of Animal Science, Faculty of Agriculture, Shahrekord University, Shahrekord, Iran
Date Added: Sun May 02 2010
Date Modified: Sun May 02 2010
Abstract: None of the procedures yielded DNA of suitable purity for SSR and other PCR assays for DNA that was stored at 4C on prolonged period. We established an improved procedure for rapid isolation of DNA from sheeps blood and other species stored at 4C for up to one year or more and suitable for SSR analysis and other PCR-based applications

Introduction

PCR-based methods are widely used in plants and animals for marker-assisted breeding and high-resolution mapping. These studies require analysis of large number of samples, thus a DNA extraction method, which is fast, inexpensive and yields high quality DNA, is desired. Several methods for extraction of genomic DNA from blood, tissue, sperm, tooth and bone have been examined and demonstrated so far. Also this subject has been developed by improvement of science [1, 2]. The quality of DNA extracted from liquid blood is not adversely affected by storage at 4C for up to 24 h [3]. Small but significant changes have been observed in metabonomic studies in samples of blood maintained at 48C for 36 h [4]. DNA has been extracted from leucocytes and on prolonged storage of whole blood at -20 and -80C, DNA yield was considerably decreased which was probably due to degeneration of the white blood cells in the storage of long period. We have now modified the method for rapid isolation of DNA from blood. The DNA is suitable for SSR analysis and other PCR-based applications.

Materials and Methods

Blood collection
Blood should be collected in EDTA-containing vacutainer tubes. As will all body fluids, blood represents a potential biohazard, thus care should be taken in all steps requiring handling of blood. If the subject is from a known high-risk category, additional precautions may be required. Blood samples can be stored at room temperature for DNA extraction within the same working day or at refrigerator for later uses.

Standard chemicals
This method uses standard chemicals that can be obtained from any major supplier; we used chemicals supplied by Sigma Co. as follow:

Procedure of DNA Extraction
Before starting DNA extraction, liquid blood venogects should be shake gently by rotating blood mixer (vortex)

  1. Pour 500 l of blood into a 1.5 ml eppendorf tube and add 1000 l of red cell lysis buffer.
  2. Shake microfuge tube gently (up to homogenizing), then spin for 2 minutes at 7000 rpm.
  3. Discard supernatant and repeat steps 1-3 two or three more times to remove hemoglobin. It is important to breakdown the pellet by vortexing and rinses it well in red blood cell lysis buffer in order to clean the white blood cells from residual of hemoglobin.
  4. Placing the tube on tissue paper for few seconds downward. Be careful from cross-contamination between different samples.
  5. Add 400 l of nucleic lysis buffer to eppendorf tube. Note: if the pellet formed, you must pipette the pellet up to dissolve it.
  6. Add 100 l of saturated NaCl (5M) and 600 l of chloroform to eppendorf tube and mix on a rotating blood mixer at room temperature then spin it for 2 minutes at 7000 rpm.
  7. Transfer 400 l of supernatant to a new 1.5 ml tube.
  8. Add 800 l of cold (-20C) absolute Ethanol and shake it gently then vortex it. DNA should appear as a mucus-like strand in the solution phase.
  9. Spin the microfuge tube for one minute at 12000 rpm to precipitate, then discard supernatant carefully and let tube be completely dried in room temperature (Place Eppendorf tube downward on the tissue paper).
  10. Add 50l of TE to it then vortex; keep eppendorf tube of DNA in 4C or -20C for later uses. We routinely use about one l per PCR reaction without adverse affects. DNA can be quantified and diluted to a working concentration at this point or simply use 1 l per PCR reaction. We expect that the yield of this procedure be 100 to 300 ng/l, DNA. Using the above method, high quality DNA samples from a sheep population were extracted for gene mapping studies.

Results

The quality and quantity of extracted genomic DNA were controlled. High quality DNA was obtained using our method. All of the samples including blood samples stored at 4C for one year and other samples stored in different conditions were able to profiling for SSR and other PCR applications. Agarose gel 0.8% was used for quality control of genomic DNA. Extracted genomic DNA by our method on Agarose gel 0.8% , SSR amplification on polyacrylamide gel 6% and Digestion of DNA with EcoR1 restriction enzyme are given in Figure 1. The yields of the DNA samples ranged from 1.0 to 3.0 g from 500l blood that was stored at 4C for one year. This amount of DNA is enough to conduct 200 to 300 PCR reactions.

Figure 1. a) Genomic DNA extracted from whole blood of the sheep using the rapid isolation method on agarose gel; Lanes 1 to 5 and 6 to 10: DNA extracted from whole blood stored at -20C and +4C for one year, respectively. b) Microsatellite PCR analysis of DNA samples isolated by the rapid isolation method on polyacrylamide gel. The DNA samples were amplified for MCM137 marker from whole blood that was stored at +4C for one year. c) Digestion of DNA samples with EcoR1 restriction enzyme.

Discussion

Extracted genomic DNA from different biological samples is used widely in medical genetic laboratories for diagnosis of genetic disease and in forensic and research centers and laboratories. In our method, genomic DNA can be extracted in the least time and with high quality and quantity by using simple materials and equipments.

Not only was high quality DNA extracted from blood that was stored at 4C, this method also worked well for extracting DNA from the other blood samples that were stored at -20C or -80C. In a workday, one person can complete DNA isolation from more than 50 blood samples using this method. This method has been routinely used to extract DNA from whole blood of sheep for PCR based applications in our laboratory but it can be used for other species such as human. This method has several advantages such as; economical spending, no need to the specialized and expensive equipments, spending little time, no need to the experimented and experienced staff and more important, DNA extraction from whole blood stored at usual fridges for long time. In this method, genomic DNA with high quality and quantity can be acquired from different biological sources such as blood, bone etc. Time of extraction of genomic DNA in our method is less than one hour.
 

Acknowledgments

The authors offer grateful thanks to Shahrekord University for financial assistance; as well as to the staff of Shuli Animal Breeding Center (SABRC, Shahrekord, Iran) for providing blood of sheep.

References

  1. Bailes,S.M., Devers,J.J., Kirby,J.D. and Rhoads,D.D. (2007) an inexpensive, simple protocol for DNA isolation from blood for high-throughput genotyping by polymerase chain reaction or restriction endonuclease digestion. J. Poultry Science 86:102106.
  2. Ali,S.M., Mahnaz,S. and Mahmood,T. (2007) Rapid genomic DNA extraction (RGDE). Protocol Online PID: 4791, http://www.protocol-online.org.
  3. Antony,H., Paul,R., Yancy,R., Nicola,T., Alice,D., Rupert,G. and Stephen,L. (2007) The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4C for up to 24 h. International Journal of Epidemiology 2008;37:i7i10.
  4. Tim,C.P. and Paul,E. (2007) The UK Biobank sample handling and storage validation studies. International Journal of Epidemiology 2008;37:i2i6. 5.Bartlett,J. and White,A. (2003) Extraction of DNA from Whole Blood. Methods in Molecular Biology, 10.1385/1-59259-384-4:29.

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