DNA AND CELL BIOLOGY Volume 9, Number 6, 1990 Mary Ann Liebert, Inc., Publishers Pp. 461-469

LABORATORY METHODS

Rapid Detection of Hypervariable Regions by the Polymerase Chain Reaction Technique RONNY DECORTE, HARRY CUPPENS, PETER MARYNEN, and JEAN-JACQUES CASSIMAN

ABSTRACT The polymerase chain reaction (PCR) technique has provided a substantial improvement for the detection and analysis of known genetic polymorphisms. Here, we describe the application of this method for the detection of variable number of tandem repeat (VNTR) sequences. With the use of unique oligonucleotide primers, flanking the repeat sequence, and the thermostable Taq DNA polymerase, the hypervariable regions 3' of the Ha-ras gene, 3' of the apolipoprotein B gene, and 5' to the joining segments of the heavy-chain immunoglobulin gene could be amplified. Alíeles up to 2,000 bp could be visualized directly on ethidium bromidestained agarose gels. Larger alíeles were seen only after traditional Southern blot analysis with an internal probe. The value of this new approach for the detection of VNTRs is illustrated in a case of paternity

dispute.

and washing conditions. The same idea has been extended by Nakamura et al. (1987) to the identification and isolation of VNTR loci in human genomic libraries. Previous methods for typing hypervariable regions were based on Southern blot analysis (Southern, 1975). However, this technique has a low resolution for small size differences between relatively large DNA fragments (Baird et ai, 1986; Boerwinkle et al., 1989). For hypervariable regions, some alíeles may remain undetected because their size difference is between 11 and 70 bp. Also, one needs at least 50 ng of high-molecular-weight DNA for an analysis with locus-specific VNTR probes in Southern blotting, which may not be available in forensic samples such as

INTRODUCTION regions (HVRs) or variable number of tandem sequences (VNTRs) provide important landmarks on the human genome. Because of their high polymorphic content, they are regularly used for gene mapping (Reeders et al., 1985), the construction of linkage maps (White et al., 1985; Donis-Keller et ai, 1987), the identification of loci which may be involved in carcinogenesis (Lothe et ai, 1988), paternity determinations (Baird et ai, 1986), and forensic medicine (Gill et al., 1985). The molecular basis for their length polymorphisms was not recognized until several alíeles of the hypervariable region associated with the insulin gene had been sequenced (Ullrich et al., 1982). Analysis of other VNTR loci indicated that the number of copies of the tandem repeats could vary from one to several hundreds, giving rise to numerous alíeles differing in length by the number of repeats (Wong et al., 1986). Jeffreys et al. (1985) demonstrated that the sequence similarity between the repeats can be used to generate an individual specific 'genetic fingerprint' which detects several hypervariable regions under reduced hybridization

Hypervariable repeat genetic

Center for Human Genetics,

University

of Leuven,

blood and

semen stains or hair roots (Gill et al., 1985; al., 1987). The polymerase chain reaction technique (PCR) can enhance and facilitate the detection of known polymorphisms, even from degraded DNA samples (Mullis et al., 1986; Impraim et al., 1987). By using appropriate flanking oligonucleotides and the thermostable Taq DNA polymerase, one can amplify a specific region of interest (Saiki et al., 1988). This technique has been used with success for

Wong

et

Campus Gasthuisberg

0&N6

Herestraat, B-3000 Leuven, Belgium. -

461

DECORTE ET AL.

462

analysis of certain DNA polymorphisms in single husperm (Li et al., 1988), single diploid cells (Jeffreys et al, 1988a), and single hair roots (Higuchi et al., 1988). Here, we describe the application of the PCR for the detection of three hypervariable regions: 3' of the Ha-ras gene (Capon et al., 1983), 3' of the apolipoprotein B gene (Huang and Breslow, 1987), and 5' to the joining segments of the heavy-chain immunoglobulin gene (Silva et al., 1987). This method has been applied with success in several paternity determinations. Our results indicate that amplification of VNTR loci allows one to detect alíeles up to 2,000 bp on ethidium bromide-stained agarose gels. The

68°C were carried out on a DNA Thermal Cycler (Perkin Elmer-Cetus Instruments). After the 25th cycle, an extra step was performed at 68 °C for 10 min to extend the tem-

tion.

Analysis of PCR products

the

man

plates completely. Amplification of the VNTR 3' of the apolipoprotein B gene was carried out in a 100-fi\ reaction containing 1 x PCR-gelatin buffer (50 mMKCl, 10 mMTris-HCl pH 8.4, 2.5 mMMgCl2, and 200 fig/ml gelatin) (Saiki et al., 1988), 200 fiM of each dNTP, 1 fig of genomic DNA, 1 fiM of each oligonucleotide primer, and 2.5 units of Taq DNA polymerase. Twenty-five cycles of denaturation for 1 min at 94°C and annealing-extension for 5 min at 60°C were PCR-based method described here could be useful for typ- applied. The last polymerization step was extended for an ing allelic variation at other hypervariable regions or additional 10 min at 60° C. VNTRs which fall in a size range suitable for amplificaSi nuclease digestion

MATERIALS AND METHODS

was

performed on 15-fil PCR prod-

ucts in 0.2 M NaCl, 0.05 M sodium acetate

pH 4.5, 1 mM and 0.5% with units of 25 ZnS04, glycerol S, nuclease DNA isolation and preparation of oligonucleotides (BRL) for 2 hr at 37°C. Ten to twenty percent of the amGenomic DNA was purified from venous blood samples pliation products were separated by electrophoresis on either manually through phenol/chloroform extraction or 2% agarose or 3% composite agarose (1% Nusieve GTG, saturated NaCl (Miller et al., 1988) or on an automated FMC, and 2% regular agarose) gels in Tris-acetate buffer nucleic acid extractor (Applied Biosystems, Foster City, and visualized directly with ethidium bromide (Maniatis et CA). The Ha-ras plasmid DNA (pbc-Nl), containing the al., 1982). The size of the different alíeles was determined human proto-oncogene Ha-ras 1 (Pulciani et al., 1982), with restriction digests of pGEM, X, and 0X174 as molecuwas purified using a slightly modified alkaline extraction lar weight markers. Amplified DNA was transferred after and (Birnboim Doly, 1979). Oligonucleotides electrophoresis to Hybond N* (Amersham) according to procedure (Table 1) were synthesized on a Cyclone DNA synthesizer the conditions recommended by the manufacturer. Oligo(Biosearch, San Rafael, CA) and used without additional nucleotide probes were end-labeled with T4 polynucleotide kinase (Pharmacia) and [>-32P]ATP (Maniatis et al., purification. 1982). Prehybridization of the nylon membranes was in 3 x SSPE (1 x SSPE is 180 mM NaCl, 10 mM NaH2P04, Polymerase chain reaction and 1 mM EDTA pH 8), 5x Denhardt's solution Amplifications of the hypervariable region 3' of the Ha- (Denhardt, 1966), 0.5% NaDodSO, at 60°C for at least 1 ras gene and the 5' region of the joining segments of the hr. Hybridization was carried out overnight with heavy-chain immunoglobulin gene (HVR-Ig) were per- 32P-labeled oligonucleotide probe in the same buffer. Fil-

formed in 100-fil reactions containing 1 x PCR-DMSO ters were washed for 15 min in 2x SSPE and 0.1% buffer (1.66 mM(NH4)2S04, 67 mMTris-HCl pH 8.8, 2 mM NaDodS04 at 65°C. Autoradiography was at room temMgCl2, 10 mM/3-mercaptoethanol, 6.7 /¿MEDTA, and 17 perature using intensifying screens and Hyperfilm-ECL /ig/ml BSA), 10% DMSO, 500 fiM of each deoxynucleo- film (Amersham). tide triphosphate (dNTP), 1 fig of genomic DNA, 1 ¡iM of each oligonucleotide primer, and 2.5 units of Taq DNA Restriction analysis of genomic DNA

polymerase (Perkin Elmer-Cetus, Norwalk, CT). Twentyfive cycles of denaturation for 1 min at 94°C and annealSix micrograms of genomic DNA was digested overnight ing-extension for 10 min (Ha-ras) or 6 min (HVR-Ig) at with the restriction endonucleases Alu I, Pvu II or Xba I (3 Table 1. Sequence 5'-Ha-ras 3'-Ha-ras HVR-Ha-ras

5-Apo B

3'-Apo B 5-Ig-JH 3-Ig-JH

of

Oligonucleotide Primers

and

Probes

5'-GAGCTAGCAGGGCATGCCGC 5-AGCACGGTGTGGAAGGAGCC

5-CACTCGCCCTTCTCTCCAGGGGACGCCA 5-GAAACGGAGAAATTATGGAGGG 5'-TCCTGAGATCAATAACCTCG 5-GGGCCCTGTCTCAGCTGGGGA 5'-TGGCCTGGCTGCCCTGAGCAG

PCR DETECTION OF HYPERVARIABLE REGIONS

463

V/fig of DNA) in the presence of 3.3 mM spermidine. Amplification of the hypervariable region 3' of DNA fragments were electrophoretically separated at 2 the Ha-ras gene

V/cm on 1.6% Tris-acetate gels for 20 hr. DNA was denaturated in 0.5 NNaOH/1.5 MNaCl for 15 min and subsequently transferred by alkaline-blotting (0.4 N NaOH) to Hybond N* for at least 5 hr. Apo B and HVR-Ig probes were generated by amplification of genomic DNA from homozygous individuals. DNA fragments were isolated by electrophoresis of 30% of the amplification mixture through 0.7% low-melting temperature agarose (BRL). Gel slices were dissolved in three volumes of water at 65°C. The Ha-ras plasmid DNA and the HVR-Ig probe were labeled with 32P by the random-priming method of Feinberg and Vogelstein (1983). The Apo B probe was radioactively labeled by a minor modification of the amplification method described before; 200 ¡iM dCTP was replaced by 50 fiC\ of [a-32P]dCTP, approximately 10% of the dissolved gel slice was used as DNA template, and the end volume of the reaction mixture was reduced to 50 /A. Filters were hybridized at 42°C overnight in 40% formamide, 50 mM sodium phosphate buffer pH 7.5, 5 mM EDTA, 0.1% NaDodS04, 0.9 M NaCl, 5x Denhardt's, 100 fig/ml salmon sperm DNA (human placental DNA for the HVR-Ig probe), and 5% dextran sulfate. Final post-hybridization washes were at 65°C for 15 min in 0.3 x SSC and 0.1 % NaDodSO, ( 1 x SSC is 0.15 M NaCl and 15 mM sodium citrate pH 7.0). Filters were exposed at -70°C to Hyperfilm-ECL film using intensifying screens.

RESULTS

Selection of oligonucleotide primers for PCR of VNTRs PCR primers were designed such that they flank the region with the tandem repeats. Oligonucleotides were tested for their uniqueness by a homology search through GenBank (release 56). Sequences of all amplification primers are given in Table 1 together with an oligonucleotide (HVR-Ha-ras) complementary to the 28-bp repeat sequence of the hypervariable region 3' of the H-ras gene.

Table 2 presents the characteristics of the three different VNTRs used in this study.

Table 2. Characteristics

Chromosome VNTR

localization

Number

of

To test the applicability of the PCR technique for the detection of VNTRs, we chose the hypervariable region 3' of the Ha-ras gene as a model system. This VNTR has a high GC-content similar to most hypervariable regions presently known. Second, because we wanted to see the different amplified alíeles directly through ethidium bromide staining in the gel, we were interested in the fragment sizes detectable by the PCR technique. Several parameters including the PCR buffer (PCR-DMSO or PCR-gelatine buffer), the number of cycles (20-30), the annealing temperature (55-68cC), and the extension time (3-6-10 min) were tested (data not shown). Variation of the number of cycles showed that the different alíeles were clearly visible after 25 cycles, and increasing the number of cycles to 30 only gave rise to the appearance of additional fragments. Similar results have been obtained by Jeffreys et al. (1988a) for the amplification of large minisatellite regions. This phenomenon can be explained by the presence of some incompletely transcribed templates at the end of the extension phase. They can act as primers in the next annealing phase and hybridize at several sites of the VNTR region because of the complementarity of the tandem repeats. This results in the generation of PCR products of abnormal length which can be visualized after a sufficient number of cycles (30). As shown, limiting the number of cycles for the amplification of hypervariable regions avoids this artefact. Other improvements were obtained by raising the annealing temperature to 68°C, extending the polymerization time to 10 min and modifying the PCR-DMSO buffer to 2 mM MgCl2 and 500 fiM dNTP. Additional PCR products were sometimes seen after hybridization with the HVR-Ha-ras probe (Fig. lb, lane 4'). These fragments were probably incompletely single-stranded templates because they could be eliminated by digestion with S, nuclease (Fig. lb, lane 4). A random panel of five nonrelated Caucasians was screened for polymorphism by the PCR technique (Fig. 1). Ethidium bromide staining of the agarose gel made most alíeles visible (Fig. la), although the larger alíeles (-2,100 bp) were less efficiently amplified similar to what was observed by Jeffreys et al. (1988a). These DNA fragments

of the

VNTRs Used

Alíele

length

in

This Study

alíeles*

ranged

Repeat length b

271-2,763 520-1,720

28 50

541-871

Ha-ras

llpl5.5

HVR-Ig Apo B

14q32

18 6

2p24-p23

12

2

x

%GC^

67 15

64 4

Amplified fragment length M 943 720 661

aThe number of alíeles and alíele lengths observed in a Caucasian population were obtained from data published for Ha-ras, Silva et al. (1987) for HVR-Ig, and Boerwinkle et al. (1989) for Apo B.

by Baird et al. (1986) bLength in bp.

CGC content of the VNTR repeat units. dAmplified fragment length was calculated after designing of oligonucleotide primers from sequence data lished by Capon et al. (1983) for Ha-ras, Silva et al. (1987) for HVR-Ig, and Knott et al. (1986) for Apo B.

pub-

DECORTE ET AL.

464

C

1

-

tflrt

a

-

2322 2027

1 2 3 4 4' 5

1353

1078

• *•

-

872

603 DNA amplification of the hypervariable region 3' of the Ha-ras gene for different individuals (lanes 1-5). The amplified alíeles are shown after ethidium bromide staining of the agarose gel (a) and Southern blot analysis with the HVR-Ha-ras oligonucleotide (b), respectively, c. Southern blot of the same individuals digested with Pvu II.

FIG. 1.

Table 3. Amplification Fragment Sizes and Allele Frequencies Observed the Three Different VNTRs

Ha-ras PCR fragment* 859 915 943 971 999

1,055 1,083 1,391 1,419 1,447 2,035 2,091 2,147 2,511 2,539 2,595 2,651

Heterozygosity (%)

HVR-Ig

for

Apo B

frequency

PCR fragment*

frequency

PCR fragment*

frequency

0.006 0.006 0.015 0.641 0.032 0.032 0.038 0.020 0.058 0.020 0.032 0.038 0.006 0.038 0.006 0.006 0.006

470 520 570 670 870 920

0.173 0.028 0.402 0.229 0.145 0.023

541 571 601 631 661 691 721 781 811 841 871

0.021 0.105 0.079 0.220 0.368 0.074 0.011 0.026 0.032 0.053 0.011

58

73

79

156

214

190

Number of chromosomes

typed

aFragment sizes were determined to the

nearest

repeat length and

are

expressed in bp.

465

PCR DETECTION OF HYPERVARIABLE REGIONS

detected after Southern blot analysis with the HVRHa-ras oligonucleotide probe (Fig. lb). To check the validity of the amplification procedure, genomic DNA of the five individuals was digested with Alu I and probed with a J2P-labeled Ha-ras gene probe (Fig. lc). After autoradiography, the same polymorphic bands were revealed as detected after amplification of genomic DNA. The Mendelian inheritance of the different alíeles was tested in several families, one of which is shown in Fig. 3a (below). In a random population of 78 persons 17 different alíeles with fragment sizes between 859 and 2651 were observed (Table were

3).

Amplification of the hypervariable region 5' the joining segments of the heavy-chain immunoglobulin gene A similar used for the

to

approach as for the Ha-ras VNTR has been amplification of the hypervariable region 5' to

the joining segments of the heavy-chain immunoglobulin gene (HVR-Ig). After 25 cycles, different fragments were visible of which some were constant among different individuals and others were polymorphic (data not shown). These additional fragments were avoided by raising the annealing temperature to 68°C but an additional band still remained on the ethidium bromide-stained gel. This band could be removed by S, nuclease digestion. A random panel of five nonrelated Caucasians was used in a search for polymorphism in this region (Fig. 2a). Southern blot analysis of genomic DNA of the five individuals digested with Pvu II and probed with a 32P-labeled amplification fragment (Fig. 2b) did show the same polymorphic fragments as after amplification. Also Mendelian inheritance was observed in several families (Fig. 3b). Six different alíeles were detected in a random population of 107 individuals (Table 3). When the amplified alíeles were compared with the expected fragments, based on the published sequence data (Silva et al., 1987; Table 2), a discrep-

1198

5

1

2

3

H

5

676

517 460

1353 1078

872

DNA amplification of the hypervariable regions 5' to the joining segments of the heavy-chain immunoglobulin (HVR-Ig) and 3' of the apolipoprotein B gene (Apo B). a and c. Amplified fragments for different individuals (lanes 1-5) size-fractionated on an agarose gel and stained with ethidium bromide, b and d. Southern blot analysis of genomic DNA of the same individuals digested with Pvu II and Xba I, respectively. FIG. 2.

gene

466

DECORTE ET AL. a

Amplification of the 3' apolipoprotein hypervariable region

DrO

Ha-ras

Dp

b

à ààà

HVR-lg c

OrQ

Apo

B

FIG. 3. Mendelian inheritance of the different alíeles detected after DNA amplification of the hypervariable regions of Ha-ras (a), HVR-lg (b), and Apo B (c). The amplified fragments were separated on agarose gels and visualized with ethidium bromide. First and last lanes are molecular weight markers.

length (one repeat unit [50 bp] less for our amplified fragments) was observed. Nevertheless the alíeles we detected with the PCR technique were identical in length to the alíeles observed on genomic blots (data not shown). Two additional alíeles (520 and 920 bp) were observed which were not detected before by Silva et al. (1987). Two rare, described alíeles (one in 186 chromosomes) were not present in our population sample. ancy in

B

The hypervariable region 3' of the apolipoprotein B gene (Apo B) is very AT-rich (Table 2) and presents a distinct family of VNTRs. These VNTRs are each unique in the human genome (Huang and Breslow, 1987) and cannot be used to detect other possible AT-rich VNTRs, even under reduced stringency of hybridization and washing. DNA amplification of this hypervariable region was tested in two different buffers (PCR-DMSO and PCR-gelatin) and at different annealing temperatures (data not shown). Optimal results were obtained in the PCR-gelatin buffer with annealing and extension at 60°C. At higher extension temperatures (68-72°C), additional bands were observed. The low GC content (less than 8%) of the amplified region probably causes the template to denature before complete

extension occurs when the temperature rises from 60° C to 72°C. Seven different amplified alíeles could be detected in a random sample of five nonrelated individuals after ethidium bromide staining of the agarose gel (Fig. 2c). A Southern blot analysis of genomic DNA of the five individuals digested with Xba I was carried out with a 32P-labeled amplification fragment (Fig. 2d) and revealed the same polymorphisms as after amplification (Fig. 2c). Because of the low GC-content of the amplified Apo B hypervariable region, we adapted the PCR procedure for labeling this fragment with [a-32P]dCTP as described in the Materials and Methods section. Mendelian inheritance of the polymorphic alíeles was determined in several families and was consistent with an autosomal codominant trait (Fig. 3c). In a random population of 95 Caucasians, 11 different alíeles were observed (Table 3). A 12th alíele (751 bp) was only seen for a child in a nonpaternity case (data not shown). During the course of this study, two papers were published describing the use of the PCR technique for the detection of the hypervariable region 3' of the apolipoprotein B gene (Boerwinkle et al., 1989; Ludwig et al., 1989). Our results are in close agreement with the data of the first group of investigators while Ludwig et al. (1989) detected two additional alíeles in a population sample much larger than the one presented here.

Application of Ha-ras, HVR-lg, and Apo B polymorphisms for paternity determinations To test the applicability of this approach for genetic identification studies, a paternity case was investigated with the Ha-ras, HVR-lg, and Apo B polymorphisms. Figure 4 presents the results of the analysis. The HVR-lg and Apo B polymorphisms exclude the presumed father (F) from being the biological father of the child (C). This result was confirmed with other VNTRs (data not shown). Although the Ha-ras polymorphism could not exclude paternity in this example, it is a hypervariable region of interest for paternity determinations because it contains several rare alíeles (Table 3). The procedure from DNA amplification until gel electrophoresis and photography could be

PCR DETECTION OF HYPERVARIABLE REGIONS

F C M

a

F

C

467

M

670 570

470

Ha-ras

HVR-ig

Apo

B

with the Ha-ras, HVR-Ig, and Apo B polymorphisms by direct visualization of the different alíeles on agarose gels. Lane F is the presumed father while lane C is the child and lane M the mother of the child. First and last lane contain molecular weight markers.

FIG. 4.

Paternity analysis

amplified

done in 1.5 days. This way, the paternity analysis was reduced from more than 1 week for a traditional Southern blot with hybridization and autoradiography to less than 2

freys et al., 1988a; Boerwinkle et al., 1989; Bowcock et al., 1989; Horn et al., 1989; Ludwig et al., 1989) increasing the number of amplifiable VNTR loci to 11, including those described here. This set of VNTR's can be expanded with days. other hypervariable regions, if they have an alíele distribution in a size range (100-2,000 bp for direct detection) suitable for amplification. Thus, this is a rapid and simple DISCUSSION method for detection of a highly polymorphic system of genetic markers. A rapid and simple procedure for the detection of hyAs demonstrated here, for paternity determinations, the pervariable regions or variable number of tandem repeat detection of polymorphic alíeles by the PCR technique can (VNTR) sequences is presented here. Making use of unique be done within 2 days. Although genetic identification oligonucleotides flanking the repeat region and the PCR studies are only one field of application, other genetic technique, reproducible and faithful amplification of the studies may benefit also from this procedure. For Ha-ras, hypervariable regions 3' of the Ha-ras gene (Capon et al., it has been reported by Kontriris et al. (1985) that the pres1983), 3' of the apolipoprotein B gene (Huang and Bres- ence of rare alíeles may be linked to susceptibility to canlow, 1987), and 5' to the joining segments of the heavy- cer. Other groups have demonstrated the use of the hyperchain immunoglobulin gene (Silva et al., 1987) has been variable region 3' of the Ha-ras gene for detection of pardemonstrated. With this method, it is possible to visualize tial deletions of chromosome lip in tumors (Ali et ai, polymorphic fragments of up to 2,000 bp directly on ethid- 1987; Mackay et al., 1988). The VNTR 5' to the joining ium bromide-stained agarose gels. Larger alíeles are only segments of the heavy-chain immunoglobulin gene (HVRseen after Southern blot analysis with an internal probe beIg) may be of interest for studies of genetic association in cause the yield of amplification product is too low for diseases of the immune system. DNA variability in the hydirect detection (Jeffreys et al., 1988a; this study). Typing pervariable region 3' of the ApoB gene could be associated hypervariable regions by the PCR technique is much more with myocardial infarction (Hegele et al., 1986). The imsensitive than Southern blot analysis (Boerwinkle et al., proved sensitivity and resolution for small size differences 1989). Small differences in length can be detected because obtained by amplification of polymorphic VNTR alíeles the primers are directly flanking the repeat region. This will enhance genetic linkage studies and gene mapping was previously not possible for VNTR loci in which the re(Skolnick and Wallace, 1988). Finally, the PCR technique striction sites were localized much further away. Another can be used for studying mutation rates of hypervariable important feature of this method is the ability to detect regions. Previously, germ-line instability has been detected polymorphic alíeles in low amounts of DNA —even de- by traditional Southern blot analysis of human pedigrees graded—as is the case for DNA samples from forensic (Jeffreys et al., 1988b; Wolff et ai, 1988). With the PCRcases. Therefore, this new approach will certainly revolubased typing method, it is possible to analyze polymortionize individual identification in forensic medicine. phisms from single diploid cells (Jeffreys et al., 1988a) or While this study was ongoing, other reports were pub- single sperm (Li et ai, 1988), permitting a much more delished of PCR-based typing of hypervariable regions (Jef- tailed analysis of mutation rates for VNTR sequences.

468

DECORTE ET AL.

ACKNOWLEDGMENTS

HIGUCHI, R., VON BEROLDINGEN, C.H., SENSABAUGH, G.F., and ERLICH, H.A. (1988). DNA typing from single hairs. Nature 332, 543-546. HORN, G.T., RICHARDS, B., and KLINGER, K.W. (1989). Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction. Nucleic Acids Res. 17, 2140. HUANG, L.-S., and BRESLOW, J.L. (1987). A unique AT-rich

We wish to thank Sabine Hermans, Peggy Servranckx, Dominique Kellens, and Kathy Merckx for their excellent technical assistance and Staf Doucet for synthesizing the oligonucleotides. This work was supported by a grant "Geconcerteerde hypervariable minisatellite 3' to the apo B gene defines a high acties" from the Belgian Government and by the Interuniinformation restriction fragment length polymorphism. J. Biol. versity Network for Fundamental Research sponsored by Chem. 262, 8952-8955. the Belgian Government (1987-1991). Peter Marynen is a IMPRAIM, C.C., SAIKI, R.K., ERLICH, H.A., and TEPLITZ, R.L. (1987). Analysis of DNA extracted from formalin-fixed, Bevoegdverklaard Navorser of the Nationaal Fonds voor paraffin-embedded tissues by enzymatic amplification and hyWetenschappelijk Onderzoek, Belgium.

REFERENCES ALI, LU., LIDEREAU, R., THEILLET, C, and CALLAHAN, R. (1987). Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia. Science 238, 185-188. BAIRD, M., BALAZS, I., GIUSTI, A., MIYAZAKI, L., NICHOLAS, L., WEXLER, K., KANTER, E., GLASSBERG, J., ALLEN, F., RUBINSTEIN, P., and SUSSMAN, L. (1986). Alíele frequency distribution of two highly polymorphic DNA sequences in three ethnic groups and its application to the determination of paternity. Am. J. Hum. Genet. 39, 489-501. BIRNBOIM, H.C., and DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. BOERWINKLE, E., XIONG, W., FOUREST, E., and CHAN, L. (1989). Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: Application to the apolipoprotein B 3' hypervariable region. Proc. Nati. Acad. Sei. USA 86, 212-216. BOWCOCK, A.M., RAY, A., ERL1CH, H., and SEHGAL, P.B. (1989). Rapid detection and sequencing of alíeles in the 3' flanking region of the interleukin-6 gene. Nucleic Acids Res. 17, 6855-6864.

CAPON, D.J., CHEN, E.Y., LEVINSON, A.D., SEEBURG, P.H., and GOEDDEL, D.V. (1983). Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302, 33-37. DENHARDT, D.T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. DONIS-KELLER, H., GREEN, P., HELMS, C, CARTIN-

HOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T.P., BOWDEN, D.W., SMITH, D.R., LANDER, E.S., BOTSTEIN, D., AKOTS, G., REDIKER, K.S., GRAVIUS, T., BROWN, V.A., RISING, M.B., PARKER, C, POWERS, J.A., WATT, D.E., KAUFFMAN, E.R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T.R., NG, S., SCHUMM, J.W., BRAMAN, J.C., KNOWLTON, R.G., BARKER, D.F., CROOKS, S.M., LINCOLN, S.E., DALY, M.J., and ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51, 319-337. FEINBERG, A.P., and VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction fragments to high specific activity. Anal. Biochem. 132, 6-13. GILL, P., JEFFREYS, A.J., and WERRETT, D.J. (1985). Forensic application of DNA 'fingerprints'. Nature 318, 577-579. HEGELE, R.A., HUANG, L.-S., HERBERT, P.N., BLUM, C.B., BURING, J.E., HENNEKENS, C.H., and BRESLOW, J.L. (1986). Apoliprotein B-gene DNA polymorphisms associated with myocardial infarction. N. Engl. J. Med. 315, 15091515.

bridization with sequence-specific oligonucleotides. Biochem. Biophys. Res. Commun. 142, 710-716. JEFFREYS, A.J., WILSON, V., and THEIN, S.L. (1985). Hypervariable 'minisatellite' regions in human DNA. Nature 314, 67-73. JEFFREYS, A.J., WILSON, V., NEUMANN, R., and KEYTE, J. (1988a). Amplification of human minisatellites by the polymerase chain reaction: Towards DNA fingerprinting of single cells. Nucleic Acids Res. 16, 10953-10971. JEFFREYS, A.J., ROYLE, N.J., WILSON, V., and WONG, Z. (1988b). Spontaneous mutation rates to new length alíeles at tandem-repetitive hypervariable loci in human DNA. Nature 332, 278-281.

KNOTT, T.J., WALLIS, S.C., PEASE, R.J., POWELL, L.M., and SCOTT, J. (1986). A hypervariable region 3' to the human apolipoprotein B gene. Nucleic Acids Res. 14, 9215-9216. KONTRIRIS, T.G., DIMARTINO, N.A., COLB, M., and PARKINSON, D.R. (1985). Unique allelic restriction fragments of the human Ha-ras locus in leukocyte and tumour DNAs of cancer patients. Nature 313, 369-374. LI, H., GYLLENSTEN, U.B., CUI, X., SAIKI, R.K., ERLICH, H.A., and ARNHEIM, N. (1988). Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335, 414-417. LOTHE, R.A., NAKAMURA, Y., WOODWARD, S., GEDDEDAHL, JR., T., and WHITE, R. (1988). VNTR (variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas. Cytogenet. Cell Genet. 48, 167-169. LUDWIG, E.H., FRIEDL, W., and MCCARTHY, B.J. (1989). High-resolution analysis of a hypervariable region in the human apolipoprotein B gene. Am. J. Hum. Genet. 45, 458-464. MACKAY, J., ELDER, P.A., PORTEOUS, D.J., STEEL, CM., HAWKINS, R.A., GOING, J.J., and CHETTY, U. (1988). Partial deletion of chromosome lip in breast cancer correlates with size of primary tumour and oestrogen receptor level. Br. J. Cancer 58, 710-714. MANIATIS, T., FRITSCH, E.F., and SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). MILLER, S.A., DYKES, D.D., and POLESKY, H.F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215. MULLÍS, K., FALOONA, F., SCHARF, S., SAIKI, R., HORN, G., and ERLICH, H. (1986). Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263-273. NAKAMURA, Y., LEPPERT, M., O'CONNELL, P., WOLFF, R., HOLM, T., CULVER, M., MARTIN, C, FUJIMOTO, E., HOFF, M., KUMLIN, E., and WHITE, R. (1987). Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235, 1616-1622. PULCIANI, S., SANTOS, E., LAUVER, A.V., LONG, L.K.,

PCR DETECTION OF HYPERVARIABLE REGIONS

469 tu-

T., and JEROMINSKI, L. (1985). Construction of linkage

REEDERS, S.T., BREUNING, M.H., DAVIES, K.E., NICHOLLS, R.D., JARMAN, A.P., HIGGS, D.R., PEARSON, P.L., and WEATHERALL, D.J. (1985). A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature 317, 542-544. SAIKI, R.K., GELFAND, D.H., STOFFEL, S., SCHARF, S.J., HIGUCHI, R., HORN, G.T., MULLÍS, K.B., and ERLICH, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. SILVA, A.J., JOHNSON, J.P., and WHITE, R.L. (1987). Characterization of a highly polymorphic region 5' to JH in the human immunoglobulin heavy chain. Nucleic Acids Res. 15,

313, 101-105. WOLFF, R.K., NAKAMURA, Y., and WHITE, R. (1988). Molecular characterization of a spontaneously generated new alíele at a VNTR locus: No exchange of flanking DNA sequence. Genomics 3, 347-351. WONG, Z., WILSON, V., JEFFREYS, A.J., and THEIN, S.L. (1986). Cloning a selected fragment from a human DNA 'fingerprint': Isolation of an extremely polymorphic minisatellite. Nucleic Acids Res. 14, 4605-4616. WONG, Z., WILSON, V., PATEL, I., POVEY, S., and JEFFREYS, A.J. (1987). Characterization of a panel of highly var-

and BARBACID, M. (1982). Transforming genes in human mors. J. Cell. Biochem. 20, 51-61.

3845-3857.

SKOLNICK, M.H., and WALLACE, R.B. (1988). Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs). Genomics 2, 273-279. SOUTHERN, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. ULLRICH, A., DULL, T.J., GRAY, A., PHILIPS, III, J.A., and PETER, S. (1982). Variation in the sequence and modification state of the human insulin gene flanking regions. Nucleic Acids Res. 10, 2225-2240. WHITE, R., LEPPERT, M., BISHOP, D.T., BARKER, D., BERKOW1TZ, J., BROWN, C, CALLAHAN, P., HOLM,

maps with DNA markers-for human chromosomes. Nature

iable ministatellites cloned from human DNA. Ann. Hum. Genet. 51, 269-288.

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