The Seoul Journal of Medicine

Vol. 32, No. 4: 21 7-221, December 1991

Amplification of VNTR Locus D l SaO in DNA Recovered from Dried Bloodstains'

Jung Bin Lee

Department of Forensic Medicine, Seoul National University College of Medicine,

Seoul 110-460, Korea

=Abstract=As a method of bloodstain testing for the identification of individuals, D1S 80 locus was analyzed in bloodstains using amplified fragment length polymorphism (AMP-FLP) analysis technique. Dried blood samples were prepared on sections of cotton shirt and glass plate with 10-y1 aliquots of blood drawn from two individuals, to simulate bloodstain evidence found a t crime scenes. After aging a t room temperature for 1 month, 3 months and 11 months, DNA was isolated and amplified with primers of DlS8O locus, followed by acrylamide gel electrophoresis and ethidium bromide staining. The amount of DNA recovered from a 10-yl dried blood aliquot was approximately 440 ng, regardless of the dried pattern or the aging. In quality, the DNA became degraded as aging was extended. The AMP-FLP profile of the bloodstain was identical to that of the peripheral blood, which was not affected by the dried pattern or the aging.

Key Words: PCR, VNTR, DIS80, Bloodstain

INTRODUCTION

Phenotype makeup determined in biological ma- terials recovered as criminal evidence has been used as a potent tool for personal identification or exclusion. Blood evidence is the most frequently encountered evidence at crime scenes. Polymor- phic proteins and cellular antigens are currently typed in forensic blood samples using electropho- retic and immunologic techniques (Sensabaugh 1981 & 1982). However, forensic blood samples are usually submitted in the form of dried stains and are contaminated. As the protein is frequently prone to be denatured by drying and contamina- tion, the drying and contamination as well as the aging frequently associated with forensic blood- stains limit the available number of protein markers.

+ This study was supported in part by research grant of Seoul National University Hospital (1987).

Since DNA is very stable in dried form, DNA ana- lysis can overcome the limiting factors of protein markers ecountered in the identification analysis of bloodstains. Furthermore, DNA provides a larger number of distinguishable alleles especially in the loci of Variable Number of Tandem Repeats (VNTR) (Baird et al. 1986; Balazs et al. 1989; Wong et al. 1986), than the currently available protein markers. VNTR analysis for individual identification has been performed on bloodstains (Gill et al. 1985; Kanter et a/. 1986) using restriction fragment length poly- morphism (RFLP) analysis technique. Although ex- tremely effective for VNTR analyses, the RFLP me- thod requires a relatively large amount of undegra- ded DNA; micrograms for multi-locus analysis (Jeff- reys et al. 1985) and hundreds of nanograms for single-locus typing (Wong et al. 1987). Usually such an amount of DNA can not be obtained from bloodstains. Using PCR (Saiki et al. 1985) only a few nanograms of human DNA have been sufficient for analyzing a certain VNTR locus (Kasai et al. 1990; Lee 1991a, b). This paper presents the

MATERIALS AND METHODS

amplification fragment length polymorphism (AMP- CGCCG-3' and 5'-GTCTTGTTGGAGATGCACGTG- FLP) of D1S80 VNTR locus in DNA recovered from CCCCTTGC-3'. Two nanograms of each sample bloodstains in order to demonstrate the feasibility were amplified in 25 p1 of a reaction mixture con- of matching the DNA profile recovered from a taining 67 mM Tris-HCI (pH 8.3), 6.7 mM magnesium bloodstain with that obtained from a blood sample chloride, 16.6 mM ammonium sulfate, 10 mM 2-me- of a suspected individual. rcaptoethanol, 170 pg/ml bovine serum albumin,

10% dimethyl sulfoxide, 2.5 mM each of the deoxy forms of adenosine triphosphate, cytidine triphos- phate, guanosine triphosphate, thymidine triphos-

Preparation of bloodstain samples: phate, 1.25 units of Taq polymerase, and 2 yM Dried blood samples were prepared to simulate of each primer. After denaturation of the DNA at

bloodstain evidence found on clothing and on a 95k for 1 min, annealing was done at 65k for smooth surface. 10-yl aliquots of freshly drawn 1 min, with an extension at 70k for 8min, and re- blood from two individuals were pipetted onto sec- peated for 30 cycles. The polymorphism of the tions of a cotton shirt producing stains approxima- amplified products was analysed by ethidium bro- tely 6 mm in diameter. Another 10-p1 aliquots were mide staining after electrophoresis on polyacryla- pipetted onto a glass plate to make a stain-dot mide gels. approximately 5 mm in diameter. These samples were allowed to age at room temperature for 1 month, 3 months and 1 1 months before DNA was isolated. One forensic bloodstain, aged two mon- ths, was taken from the cotton pants of the male suspect in a rape case. Blood was also drawn from the victim and the suspect.

Isolation of DNA: DNA was isolated from the peripheral blood as

described by Kunkel et a/. (1982). The bloodstained cotton cloth was cut into small strips with scissors, and dried blood was scraped from the glass plate with a knife blade. Both specimens were suspen- ded in DNA lysis buffer containing 10 mM Tris-HCI pH 7.4, 10 mM sodium chloride, 10 mM ethylene- diaminetetraacetate (EDTA), 100 pglml proteinase K, and 1% sodium dodecylsulface (SDS). Samples were incubated at 37k overnight with gentle mix- ing. Then the solutions were extracted twice with buffered phenol, once with phenol/chloroform (1 : 1) and once with chloroform, followed by alcohol precipitation. DNA concentrations were determined by DNA fluorometer (TKO 100 Fluorometer, Hoeffer Scientific Instruments).

RESULTS

The amount of DNA recoverable from 10 p1

1 2 3 4 M

Amplification and electrophoresis: Amplification of D1S80 was achieved using the

primers described by Kasai et al. (1990). The pri- mers were 5'-GAAACTGGCCTCCAAACACTGCC-

Fig. 1. DNA pattern isolated from bloodstains aged for 1 month (lane 2), 3 months (lane I), and 11 months (lane 3 and 4). M; size standards from lamda DNA digested with Hind Ill and Eco RI.

Fig. 2. Comparison of D1S80 AMP-FLPs between peripheral blood and 1 1 -month-aged blood- stain. The AMP-FLP of 11-month-aged blood- stain (lane 3 and 4) is identical to that of peri- pheral blood (lane 1 and 2). Lane 1, peripheral blood of individual 1; Lane 2, peripheral blood of individual 2; Lane 3, 11-month-aged blood- stain of individual 1; Lane 4, 1 1-month-aged bloodstain of individual 2; MI, 123 bp DNA ladder; M2, size maker made by author.

of dried blood stains varied from 350 ng to 650 ng in both forms dried on the sections cotton shirt and on glass plate. Aging of the bloodstain did not affect the recovery of DNA. The average reco- very was 440 ng, similar to that isolated from peri- pheral blood.

In quality, DNA recovered from bloodstains was more or less degraded; at 1 month a considerable amount of DNA larger than 21 kb could be frac- tionated by 0.7% agarose gel, but a scanty amount was fractionated at 3 months and no higher mole- cular weight DNA than 21 kb was demonstrable at 1 1 months as shown in Fig. 1.

In amplification AMP-FLP of the bloodstain DNA was identical to that of peripheral blood even in the I I-month-aged bloodstains as shown in Fig. 2. In forensic sample the AMP-FLP of the blood- stain DNA in the pants of the male suspect was consistent with that of the peripheral blood DNA of the female victim as shown in Fig. 3.

Fig. 3. D1S80 AMP-FLP analysis of a rape case. D1S 80 AMP-FLP profile of bloodstain DNA in the pants of the male suspect (lane 3) is identical to that of the peripheral blood of the female victim (lane 1). Lane 2, male suspect; MI, 123 bp DNA ladder; M2, size marker made by au- thor.

DISCUSSION

Currently a wide range of polymorphic protein markers are examined in blood including red cell enzymes, red cell antigens, and serum proteins. Of these, only a few markers have been used for bloodstain analysis; phosphoglucomutase, erythro- cyte acid phosphatase, glyoxylase I isoenzymes, group-specific component, haptoglobins, and ABO, MNSs and Rh antigen systems (Sensabaugh 1981 and 1982). Since many of the protein markers de- grade within weeks (Denault et al. 1980), they are not so suffidiently suitable as to be used commonly in bloodstain analysis. There have been some at- tempts to analyze HLA antigens in bloodstains, the most polymorphic protein system currently known (Nelson et a/. 1983). As the method requires intact lymphocytes, only a few HLA antigens have been typed successfully in bloodstains.

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As with the advent of RFLP technique in DNA analysis, several probes have been developed to analyze human genomic DNA and some have been applied to forensic samples for individual identification (Gill et a/. 1985 and 1987; Giusti et al. 1986; Honma et a/. 1989; Kanter et al. 1986). Accordingly, discrimination potential has increased remarkably by using the RFLP technique in blood- stain analysis. But the RFLP method using the Southern blot technique is time consuming and costly, and a radioisotopic assay is necessary to achieve an appropriate sensitivity. Furthermore, it requires a large amount of DNA compared to the PCR method. Kanter et al. (1986) reported that sufficient amount of blood would be as large as 100 p1 for one RFLP bloodstain analysis. In con- trast, the amount of DNA required for PCR is quite small. In this paper D1S80 locus was successfully amplified and analyzed with only 2 nanograms of human genomic DNA. Even in a single sperm two genetic loci have been analyzed using PCR (Li et al. 1988).

The alleles associated with the D1S80 locus are resolved into discrete entities using the AMP-FLP analytical technique (Budowle et al. 1991; Kasai et al. 1990), and 21 alleles with their frequencies are reported in Caucasian and Japanese indivi- duals (Kasai et al. 1990). Analysis of hudreds of unrelated individuals has generated a statistical database of allele frequencies for the D1S80 locus, in which 37 alleles have been detected (data will be reported elsewhere).

Although the D1S80 profile of the victim is identi-

cal to that of DNA isolated from the suspect's pants in the forensic sample, we can not say with these findings that the blood dried on the pants was drawn from the victim. In order to apply these find- ings to forensic identification, the allele frequencies and/or phenotype frequencies should be investiga- ted in the corresponding ethnic group or population for calculation of probability of identity. The discri- mination potential of a genetic locus relies on the number and frequency of the observed alleles. In this aspect, the AMP-FLP analysis of D1S80 can be a useful and powerful tool for identity investiga- tion, since its alleles are numerous and rather wi- dely distributed.

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