Rapid Extraction of High Quality DNA from Whole Blood Stored at 4ºC 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: firstname.lastname@example.org 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 4ºC on prolonged period. We established an improved procedure for rapid isolation of DNA from sheep’s blood and other species stored at 4ºC for up to one year or more and suitable for SSR analysis and other PCR-based applications|
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 4ºC for up to 24 h . Small but significant changes have been observed in metabonomic studies in samples of blood maintained at 48°C for 36 h . DNA has been extracted from leucocytes and on prolonged storage of whole blood at -20 and -80ºC, 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 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.
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)
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 4ºC 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 µg from 500µl blood that was stored at 4ºC 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 -20ºC and +4ºC 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 +4ºC for one year. c) Digestion of DNA samples with EcoR1 restriction enzyme.
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 4ºC, this
method also worked well for extracting DNA from the other blood samples that
were stored at -20ºC or -80ºC. 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.
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.