Extraction of DNA from the whole blood by silica gel.

Introduction

The traditional procedure for blood DNA isolation is laborious and time- consuming. It comprises separation of white cells' fraction from the whole blood, lysis with detergent, digestion with proteinase, extraction with phenol and precipitation of DNA with alcohol.

Being applied to human tests such complicated procedure leads to elevated risk for laboratory personnel to be infected by AIDS, hepatitis, cytomegalovirus as well as the other viruses potentially present in blood. In this respect an approach seems to be very attractive to treat the whole blood with such a reagent that simultaneously lyses the blood and denatures the viral particles. Use of the strong chaotropic agent GuSCN for lysis of human cells followed by sorbtion of DNA to glass powder was described by Boom (1) and Bush (2). This approach employs the ability of glass-based sorbents to bind nucleic acids at high salt concentrations and release at low ones.

The great variety of glass sorbents has been reported last years to be used for DNA extraction from various sources, containing nucleic acids. For reference, isolation of DNA fragments from agarose gel (4), plasmid DNA from E.coli (3) and genomic DNA from eukariotic and prokaryotic cells (1) could be brought up. Nevertheless, the whole blood represents a certain difficulty for immediate DNA extraction due to presence of large amount of protein in the sample. Thus, white cells containing DNA count for only about 2% of human blood cells. This was the reason that DNA extracts appeared to be contaminated by red material, inhibiting PCR amplification, when we tried to apply available glass based DNA isolation procedures directly to the whole blood.

However, developing an automated device for blood DNA extraction we preferred the glass-based biochemical concept, what enabled to perform the whole procedure in one tube. Optimized parameters of the procedure, improved composition of washing solutions together with the new choice of the glass sorbent resulted in the new short and inexpensive protocol which provides DNA of perfect quality for enzymatic reactions and PCR analysis.

Reagents

• The composition of GUANIDINE SOLUTION was: 6 M GuSCN, 20 mM EDTA, 10 mM Tris-HCl (pH 6.5), 40 g/l Triton X-100 and 10 g/l DTT.
• BIND MIX was prepared by resuspending 4 g of silica gel (Aldrich, #28,851-9) in 100 ml of GUANIDINE SOLUTION.
• PROPANOL WASH contained of 25% iso-propanol, 25% ethanol, 100 mM NaCl and 10 mM Tris-HCl (pH 8.0). All reagents can be stored at room temperature.

Procedure

1. Mix in 2.2 ml minicentrifuge tube 0.5 ml of whole blood (containing 50 mM EDTA as anticoagulant) with 1.0 ml of BIND MIX.
2. Incubate at room temperature for 3 min.
3. Collect silica gel by short spin (3 sec, 5000 g).
4. Resuspend the pellet in 1.0 ml of GUANIDINE SOLUTION by vortexing.
5. Pellet the sorbent by short spin (at 5000 g).
6. Repeat washing the silica gel with GUANIDINE SOLUTION.
7. Then, wash the silica gel with PROPANOL WASH (twice) and pure ethanol (once) in the same way.
8. Thoroughly remove ethanol and dry the silica gel under vacuum with heating applied.
9. Resuspend the silica gel in 100 ul of TE buffer. Elute DNA at 65oC for 3 min.
10. Resuspend the mix again, centrifuge for 10 sec, take the supernatant.

Time, yield and quality

Parameters of the procedure

The following new elements have been introduced in the procedure in order to provide maximal yield and quality of DNA and get rid of contaminants in the product:
1. The silica gel Aldrich (#28,851-9) was selected as a result of comparative testing of commercially available glass-based sorbents.
2. Chaotropic salt guanidine thiocyanate was used in combination with nonionic detergent Triton X-100 at 4% concentration and anti-oxidizing agent DTT at 1% concentration in GUANIDINE SOLUTION.
3. Iso-propanol and NaCl were included in the composition of alcohol washing solution.
4. The parameters of the procedure were systematically optimized to provide said requirements.

Temperature

If the blood probes are coagulated, the red clots can be preliminary dissolved in GUANIDINE SOLUTION at 65oC. However sorbtion and washing of DNA can not be performed at elevated temperature inasmuch as heating of blood-guanidine- silica gel mix leads to partial cleavage of DNA. At the same time heating doesn't notably affect binding of DNA to silica gel.

Washing and drying

The special care should be taken of preventing cross-contamination of the samples during drying under vacuum. Drying the tubes on 65oC uncovered solid-state heater is also available.

Elution

Thus, the whole procedure can be fulfilled in short time in one tube in manual mode or in simple disposable plastic cassette in automated or semi-automated mode.

The presented procedure seems to be the most appropriate for use in clinics for multiple PCR-based diagnostic tests due to its rapidity, simplicity and safety.

References

1. Boom,R., C.J.A. Sol , M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim van Dillen and J. van *der Noordaa. 1989. Rapid and Simple Method for Purification of Nucleic Acids. J. Clin. Microbiol. 28:495-503.
2. Bush,C. and M. Harvey. 1991. Rapid isolation of genomic DNA from whole blood to borosilicate particles. Clin. Chem. 37:1060.
3. Marko,M.A., R. Chipperfield and H.C. Birnboim. 1982. A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal. Biochem. 121:382-387.
4. Vogelstein,B. and D. Gillespie. 1979. Preparative and analytical purification of DNA from agarose. Proc.Natl.Acad.Sci USA. 76:615-619.
5. Maxim V.Myakishev*, George I.Kapanadze, Gadji O.Shaikhayev, Svetlana auMalahova, Georgii P.Georgiev and David R.Beritashvili "Use of perchlorate precipitation to improve plasmid isolation" Institute of Gene Biology, Moscow, 1995