Pergamon

0145-2126(94)E0019-6

Leukemia Research Vol. 18, No. 6, pp. 469--471, 1994. Elsevier Science Ltd

Printed in Great Britain 0145-2126/94 $7.00 + 0.00

LETTER TO THE EDITORS

NON-TOXIC DNA EXTRACTION IN A CLINICAL SETTING

(Received 6 December 1993. Accepted 15 January 1994)

M O L E C U L A R BIOLOGY has become an extremely use- ful tool for the detection of heritable abnormalities, and the diagnosis of neoplasms. The first step in carrying out such tests is usually the isolation of high molecular weight DNA from blood, bone marrow, tissue or cultured mononuclear cells. In a clinical setting this can mean extracting DNA from cases where the patient's blood or bone marrow is hypo- cellular, or where the availability of blood or bone marrow is limited [1]. The standard technique for DNA extraction involved either phenol or phenol : chloroform extraction of proteins [2, 3]. This process involved at least two tedious aqueous- organic phase separation steps that could allow oper- ator error. Added to this tedium was the necessity to use the toxic chemical phenol or phenol : chloroform : isoamyl alcohol for the extrac- tion, two disadvantages that encouraged the search for easier, safer alternatives.

In 1988, Miller et al. [4] reported a method of precipitating proteins selectively, using only a sodium chloride solution. A single centrifugation step can pellet the proteins precipitated by a saturated (6 M) solution of NaC1, leaving a supernatant that can be poured off to allow DNA precipitation by the addition of 100% ethanol. This technique has already been used in various clinical studies [5-8] and further modifications have also been added to help improve the yield and speed of preparation [%11]. Miller et al. [4] reported that comparable yields of DNA could result from the phenol and 6M NaCI techniques. We have compared the two techniques by digesting normal peripheral blood mononuclear cells and cal- culating the amount of DNA freed from the cells and

Abbreviations: ALL , acute lymphoblastic leukaemia: DNA, deoxyribonucleic acid; EDTA, ethylenediamine- tetraacetate; JH, immunoglobulin heavy chain joining region; MgCl2, Magnesium chloride; NaCl, sodium chloride; O.D., optical density; p, probability; PCR, poly- merase chain reaction; T:E, Tris:EDTA, (pH7.5 or pH 8.0); Tris, Tris (hydroxymethyl) aminomethane.

Correspondence to: Dr Emer Lawlor, Haematology Dept, Sir Patrick Dun's Research Laboratories, T.C.D Medical School, T.C.D., Dublin 2, Ireland.

469

its purity. Here we suggest that a slightly higher yield of DNA may be recovered by the 6 M NaC1 method, and that this DNA may be of greater purity. As a result, fewer cells are required to produce sufficient DNA to be used for PCR or Southern blot analysis.

We isolated mononuclear cells from normal vol- unteers or buffy coat preparations from normal blood donors, obtained from the Blood Transfusion Service Board, by the standard buffy cell layer separation [12] using Ficoll separation (Lymphoprep) and calcium- and magnesium-free Hanks medium (Gibco) supplemented with 0.8 g EDTA/500 ml. Cell concentrations were calculated using the ethidium bromide/acridine orange staining under U.V. light of a 1:20 dilution of cell solution in a Neubauer haematocytometer [13]. Cells were made up in ali- quots of 1.75, 2, and 2.25 million cells and were processed in pairs of equal cell numbers.

High molecular weight DNA was extracted from peripheral blood mononuctear cells by a variation of the 6 M NaC1 technique of Miller et al. [4] using overnight pronase E (Sigma) protein digestion at 37°C. The DNA was spooled out with a wire loop and dissolved in T: E (10: 1, pH 8.0). DNA concentration was estimated as ten times the optical density at 260 nm of a 1 : 200 dilution. For the phenol extraction, the NaCI protein precipitation was replaced by two × 1 min phenol : chloroform : isoamyl alcohol washes (25:24:1) saturated in 10raM Tris, pH8.0, l mM EDTA (Sigma)) [3].

The mean DNA yields of both techniques are shown in Fig. 1. Statistical analysis was performed by a Chi squared test and significance was set at a level o fp < 0.05. Although the 6 M NaCI technique appears to give better DNA yields, the mean popu- lations could not be separated significantly by Chi squared analysis (p >0.1) . Under the conditions described here, the NaCI technique appeared to yield DNA of superior purity as defined by the ratio of optical density at 260 nm/280 nm (1.7-2.0) (data not shown), although they were statistically undis- tinguishable. Warren et al. [6] showed that a variation of the Miller et al. [4] technique could yield DNA

470 Letter to the Editors

=k v

<

30

20

10

n = 30

Phenol-extracted

¢. NaCI-extracted

o i i i

0 . 5 1 .0 2 . 5 3 . 0 i

1 .5 2 . 0

:A

10 gg DNA

Millions of cells

FIG. 1. DNA isolation from low cell numbers of normal peripheral blood mononuclear cells, performed with sodium chloride or phenol:chloroform:isoamyl alcohol protein extraction (Sigma). Extractions were performed in pairs of equal cell numbers. Bars indicate standard errors.

B

4 5 6 7 8 9 1 0

: i

::!:2': i

FIG. 2. (A) Southern blot analysis with NaCl-extracted DNA. Immunoglobulin heavy chain gene rearrangement status was detected with a biotinylated JnDNA probe. Lane 1 shows biotinylated lambda HindlII-digested size marker DNA, and lanes 2-10 show 10 ~tg control (poly- clonal) or patient DNA samples digested to completion with HindlII and BamH1 restriction enzymes. Lane 2 shows control polyclonal DNA, lanes 3-5 and 8-10 show precursor-B-ALL patient DNA, lane 6 shows AML patient DNA, and lane 7 shows T-ALL patient DNA. Data and protocol as previously described [8]. (B) Polymerase chain reaction amplification of T-cell receptor gamma gene rearrangements with previously described DNA primers [16] ( 'outer ' primers only). Lane 1 shows 123 bp DNA size ladder (BRL), and lanes 2-5 show DNA from normal peripheral blood mononuclear cells amplified over 30 cycles (upper lanes) or 35 cycles (lower lanes) with varying MgC12 concentrations (2.5-5.5 mM MgCI2) (details of protocol

available from authors).

Letter to the Editors 471

which allowed the study of >700 kb D N A fragments from rare-cutting restriction enzymes by pulse field electrophoresis and Southern blotting. This com- pares with fragments of between 100 and 300 kb normally produced by phenol-based techniques [2, 3]. It is certainly suitable for D N A analysis by Southern blotting or PCR (Fig. 2).

Thus, although a statistically significant difference could not be shown between the D N A yields pre- sented here, the non-organic technique appeared to yield greater quantities of DNA~ especially from small cell numbers most often found in clinical samples. This would be of great value when the pat ient 's blood or bone marrow is hypocellular, when the availability of blood or bone marrow samples is limited, or when specific populations of cells present in small numbers are being enriched by FACS-sorting or ant ibody-conjugated magnetic beads [l, 14, 15]. Aside from this, the 6 M NaC1 technique is advan- tageous in terms of opera tor and disposal safety, since there is no need to use phenol or other organic solvents [4], and we would~ therefore, recommend it for use in clinical settings.

Acknowledgements--We thank the Blood Transfusion Service Board for blood samples, Dr Mark Lawler for helpful discussion and Dept Radiation Oncology, Glasgow University for use of their facilities. This work was sup- ported by grants from the Health Research Board, and the Childrens Leukaemia Research Project, Ireland.

References

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2. Blin N. & Stafford D. W. (1976) A general method for isolating high molecular weight DNA from eukaryotes. N.A.R. 3, 2303.

3. Sambrook J., Fritsch E. F. & Maniatis T. (1989) Mol- ecular Cloning, A Laboratorv Manual, 2nd Edn. Cold Spring Harbor Lab. Press, Cold Spring Harbor.

4. Miller S. A., Dykes D. D. & Polesky H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. N.A.R. 16, 1215.

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12. Boyum A, (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. lnvest. 32, (Suppl. 97), 77.

13. Lee S. K., Singh J. & Taylor R. B. (1975) Subclasses of T-cells with different sensitivities to cytotoxic anti- body in the presence of anesthetics. Eur. J. bnmun. 5. 259.

14. Harst D. van der, Jong D. de, Limpens J., Kluin P. M., Rozier Y., Omman G. J. B. van & Brand A. (1990) Clonal B-cell populations in patients with idiopathic thrombo-cytopaenic purpura. Blood 76, 2321.

15. Leivestad T., Gaudernack G., Ugelstad J., Vartdal F. & Thorsby E. (1987) Isolation of pure and functionally active T4 and T8 cells by positive selection with anti- body-coated monosized magnetic microspheres. Transpl. Proc. 19, 265.

16. Bourguin A., Tung R., Galili N. & Sklar J. (1990) Rapid, non-radioactive detection of clonal T-cell recep- tor gene rearrangements in lymphoid neoplasms. Proc. natn. Acad. Sci. U.S.A. 87, 8536.

GERALD MARTIN and EMER LAWLOR Haematology Department

T.C.D. Medical School T.C.D., Dublin 2

Ireland