Methanol for preparing hair bulbs for PCR - BioMedSearch

.=) 1992 Oxford University Press

Nucleic Acids Research, 1992, Vol. 20, No. 23 6419-6420

Methanol for preparing hair bulbs for PCR Chun-Ya Han, Bruce K.Lin and Henry J.Lin* Division of Medical Genetics, Department of Pediatrics, Harbor-UCLA Medical Center, 1124 W. Carson Street, Torrance, CA 90502, USA Received October 16, 1992; Accepted November 2, 1992 Hair bulb DNA, used in crime labs, is also a source of DNA for population surveys or family studies. DNA typing from single hairs is possible with the polymerase chain reaction (PCR; reference 1). Hair bulbs are usually washed in ethanol and digested with Proteinase K, and the DNA is extracted with phenol/chloroform (2). We found, during our population survey of polymorphic N-acetyltransferase (NAJ2) alleles, that hair bulbs can be fixed in methanol and used directly in PCR. Two bulbs cut from freshly plucked hairs were placed in a PCR tube (MicroAmp, Perkin Elmer Cetus) and fixed by covering them with 10 ,tl of methanol (Fisher,A.C.S. certified) and letting the methanol evaporate at room temperature over several hours. Scissors used to cut the hairs were wiped with 70% ethanol between samples to avoid cross-contamination. 13.5 j1l of buffer containing one pair of primers was added to the methanol-treated specimens. Final concentrations were 50 mM Tris-HCl pH 9.5, 20 mM (NH4)2S04, 1.5 mM MgC12, 40 AM of each deoxynucleotide triphosphate, and 1 AM of each primer. One drop of mineral oil (15 Ml) was added, and the tube was heated to 950 (all temperatures in 0C) for 15 min in a Perkin Elmer Cetus Model 9600 thermal cycler. The temperature was lowered to 850 for 10 min, and 0.25 U of Taq polymerase (1.5 Al; Promega) was added. Amplification conditions were 40 sec at 540 and 1 min at 720 followed by 30 cycles of 940 (30 sec), 540 (40 sec), and 720 (1 min). The temperature was held at 720 for 5 min after the last cycle. DNA fragments were detected by electrophoresing 13-14 M1 of PCR product on a 3% NuSieve:agarose (1:1 with 0.8 ,g/ml of ethidium bromide; 10 cm long) gel at 80 V for 30 min. Thirty-two out of 35 (91 %) methanol-fixed hair bulb samples (collected from 14 individuals) gave detectable PCR products, whereas only nine out of 37 (24%) unfixed hair bulb samples showed PCR products (examples in Figure 1). The amount of product did not appreciably increase as the number of hairs was increased from two to six (not shown). The intensity of the signal was comparable to that obtained with DNA from dried blood on blotter paper (1 x 1 mm square of dried blood in a 15 Ml PCR; Figure 2). We were able to amplify a 697 bp fragment, but not a 916 bp fragment (not shown). PCR amplification of hair bulbs fixed in methanol was not as reliable as PCR from dried blood spots, but the success rate compared favorably to that obtained with hair bulbs prepared by standard microextraction methods (78% in reference 3). Amplification may vary from bulb to bulb, but the size of the bulb did not correlate with failure to amplify. The exact role of *

To whom correspondence should be addressed

A 1 2 3 4 5

G

B m 6 7

H I

C I

8 9

m I 101112

I

I I 2526272A9Q3'1 32 3R34.3TS I

D

E

F

I I m 13 14 t5 16171819202122 232

J m I I 373 3qA4041 49

K

L I

r

Aq AAACAAA7AR

Figure 1. Examples of PCR on hair bulbs prepared with or without methanol. A-L represent 12 different individuals. Primers were 5'-CCAAAACCTGGTGATGG-3' (left) and 5'-CACTCTGCTTCCCAAGAT-3', which amplify DNA from positions 841 to 1126 of the NA72 gene. The left primer is specific for the wild type sequence and will not lead to a PCR product if the known G to A mutation at position 857 is present (5). For example, individual E is homozygous for the 857A mutation, so there is a no PCR product with or without methanol. The first two lanes for each individual are from hair bulbs fixed with methanol. The last two lanes for each individual are from hair bulbs not fixed with methanol.

the methanol is not known, but in blood spots it appears to fix hemoglobin (4). The ease with which they can be collected and fixed in methanol makes hair bulbs a suitable source of DNA for epidemiologic studies. They may also be useful for quickly rechecking the source of previously collected DNA in genetic linkage studies. Hair may be a simple alternative when blood cannot be readily obtained.

ACKNOWLEDGMENTS H.J.L. gratefully acknowledges support from a March of Dimes Basil O'Connor Starter Scholar Award (# 5-FY91-0567, # 5-FY92-1216), the Cancer Research Foundation of America, University of California Cancer Research Coordinating

6420 Nucleic Acids Research, 1992, Vol. 20, No. 23

Flgure 2. Detection of three different mutations in the NA72 gene by allele specific PCR on hair bulbs fLxed with methanol (panels A and C) and on dried blood spots (panels B and D). Panels A and B represent an individual who has C at position 481, G at 590, and G at 857. Panels C and D represent another individual, who has C at 481, G and A at 590, and G at 857. PCR, electrophoresis, staining, and photography were done under the same conditions for all specimens.

Committee funds, an American Cancer Society grant to the Jonsson Comprehensive Cancer Center at UCLA, and the Harbor Collegium.

REFERENCES 1. Higuchi,R., von Beroldingen,C.H., Sensabaugh,G.F. and Erlich,H.A. (1988)

Nature 332, 543-546.

2. Gill,P., Jeffreys,A.J. and Werrett,D.J. (1985) Nature 318, 577-579. 3. Westwood,S.A. and Werrett,D.J. (1990) Forensic Science International 45,

201-215. 4. Jinks,D.C., Minter,M., Tarver,D.A., Vanderford,M., Hejtmancik,J.F. and McCabe,E.R.B. (1989) Hum. Genet. 81, 363-366. 5. Ohsako,S. and Deguchi,T. (1990) J. Biol. Chem. 265, 4630-4634.