GENOMICS
7,331-334(1990)
Alumorphs-
Human DNA Polymorphisms Detected by Polymerase Chain Reaction Using A/u-Specific Primers
DANIEL SINNETT, JEAN-MARC G&&ique
DERAGON, LOUISE R. SIMARD, AND DAMIAN
LABUDA
Mgdicale, Centre de Recherche Hdpital Ste-Justine, Universit4 de Montreal, 3 175 C&e Ste-Catherine, Mont&al, Quebec, Canada H3T 1C5 ReceivedJanuary
31, 1990
itation can be overcome by PCR amplification of anonymous DNA segments that are flanked by already characterized repetitive sequences serving as priming points for the polymerization reaction. If polymorphic DNA fragments are revealed, this would also provide an opportunity to analyze multiple loci in a single PCR experiment. Here, we propose such a system of PCRamplified markers using as PCR primers oligonucleotides complementary to Ah sequences that are ubiquitous in the human genome (Deiniger et al., 1981; Jurka and Smith, 1988; Britten et al., 1988; Labuda and Striker, 1989). Since more than one-third of the approximately 700,000 copies of Ah repeats occur in closely spaced pairs, separated by less than 1 kb of single-copy DNA (Moyzis et al., 1989), more than 100,000 Alu-flanked DNA segments that are of a length readily amplified by PCR may be expected. We use the term “alumorphs” to describe genomic polymorphisms revealed by PCR amplification of Alu-flanked DNA segments. Alumorphs could result from several mechanisms including insertion or deletion of flanking Ah elements and, if PCR amplification is preceded by restriction enzyme digestion, from the presence of restriction site polymorphisms occurring within the DNA segments to be amplified (Fig. 1). In this study, we show that PCR amplification of human DNA samples using Alu-specific primers can be used to detect alumorphs and permits simultaneous analysis of multiple polymorphic loci in a single experiment.
The simultaneous analysis of multiple loci could substantially increase the efficiency of mapping studies. Toward this goal, we used the polymerase chain reaction to amplify multiple DNA fragments originating from dispersed genomic segments that are flanked by Ah repeats. Analysis of different human DNA samples revealed numerous amplification products distinguishable by size, some of which vary between individuals. A family study demonstrated that these polymorphic fragments are inherited in a Mendelian fashion. Because of the ubiquitous distribution of Ah repeats, these markers, called “alumorphs,” could be useful for linkage mapping of the human genome. A major advantage of alumorphs is that no prior knowledge of DNA sequence of marker loci is required. This approach may find general application for any genome where interspersed repetitive sequences are found. o ISBOAcademic Pan, IUC.
INTRODUCTION
Current techniques for mapping the human genome (White and Lalouel, 1988) exploit DNA sequence variation affecting recognition sites of restriction endonucleases (Botstein et al., 1980), as well as DNA length polymorphism due to allelic differences in the number of tandemly repeated simple sequence motifs (Nakamura et al., 1987). The resulting RFLP (Botstein et al., 1980) and VNTR (Nakamura et al., 1987) markers are routinely detected by the technique of Southern (1975). The polymerase chain reaction (PCR) (Saiki et al., 1985) provides an alternative, more direct approach to detecting DNA polymorphisms (Jeffreys et al., 1988; Roberts et al., 1989a). One advantage of PCR detection is its ability to reveal DNA sequence variation that may be undetectable by the technique of Southern (Weber and May; 1989, Litt and Luty, 1989; Roberts et al., 198913).This application, however, has been limited to marker loci of known DNA sequence. This lim-
MATERIALS
AND
METHODS
DNAs and Oligonucleotides Genomic DNA was purified from blood samples and digested with restriction endonucleases as previously described (Sinnett et al., 1988). Digested and nondigested genomic DNAs were diluted at 25 pg/ml with sterile deionized water. The primers for PCR were synthesized using the Gene Assembler (Pharmacia) and were used without further purification. The oligo331 All
Copyright 0 1990 rights of reproduction
osss-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
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SINNETT LOCUS 2
LOCUS 1
Alleles
BstX I
Genotypes 1
+I+
+/o
o/o
LOCUS 2
+I+
+I0
o/o
Locus
AL.
Tris-HCl, pH 8.4; 50 mM KCl; 2.5 mM MgCl,; 1 m&f DTT; 0.01% gelatin; 250 PLMeach dGTP, dATP, and TTP; 50 PLM dCTP (Pharmacia); 20 &i [32P]dCTP (3000 Ci/mmol) (NEN-DuPont). All PCR assayswere carried out in an automatic thermal cycler (PerkinElmer/Cetus) under the following conditions optimized for the primer used: 7 min denaturation at 94°C and 25 cycles of denaturation at 94”C, 30 s; annealing at 6O”C, 45 s; extension at 72”C, 60 s; completed by a final extension cycle at 72’C for 7 min. All samples were treated for 2 h with 300 pg/ml proteinase K and then passed through a Sephadex G-50 spun column to eliminate unincorporated nucleotides.
Electrophoresis
Alumorphs
:
ET
yq
FIG. 1. Schematic presentation of alumorph detection. Genomic sequences flanked by Alu elements (hatched arrows, arrowhead indicating oligo(A) tail) can be amplified using PCR primed with Aluspecific oligonucleotidee (small horizontal arrows). The pattern of amplified DNA fragments depends on the orientation and the sequence of the flanking Ah repeats. In this example, Ah pairs are shown in a tail to tail orientation and a single primer directs amplification of intervening DNA sequences. When both Alu repeats are present, an amplified product is generated (‘I+” allele, locus 1). In the absence of one repeat, no amplification occurs (“0” allele, locus 1). In turn, restriction enzyme digestion of DNA samples prior to amplification reveals additional polymorphisms since DNA segments containing the restriction site cannot be amplified In locus 2, the absence of an internal B&XI site permits amplification (,,+” allele), while the presence of the BstXI site precludes amplification (“0” allele). Fractionation of the reaction products allows for simultaneous analysis of different loci, each characterized by a given DNA fragment length. The absence of a fragment indicates homozygosity for the null allele (O/O), whereas the presence of the PCR product demonstrates either homozygosity for the dominant allele (+/+) or the heterozygous state (f/O). Distinction between +/+ homozygotes and +/O heterozygotes requires quantitative analysis. Since not all Ah elements are identical, but are divided into families (4,7) with each member of a family accumulating mutations independently (9), the sequence specificity of primers controls the number of DNA segments amplified In addition to the illustrated polymorphisms, a small number of amplifiable fragments may be expected to exhibit length polymorphisms due to insertions or deletions of the intervening DNA.
nucleotide primer used, $GGTGAAACCCCGTCTCTACTAAAS’, corresponds to positions 101-123 of the young Alu family consensus (Labuda and Striker, 1989).
Amplification of Alumorphs Chain Reaction
Using the Polymerase
The PCR was carried out in a total volume of 50 ~1 with 50 ng of genomic DNA, 1 &f primer oligonucleotide, and 1 unit Taq polymerase (BRL) in 40 mM
The [32P]dCTP-labeled reaction products were fractionated on a 7 or a 9% polyacrylamide gel (20 X 40 cm, 0.4-mm thickness) in TBE buffer (89 mM Trisborate, 89 mM boric acid, 8 mM EDTA) under nondenaturing conditions. A total of 25,000 cpm from each sample were dried under vacuum, resuspended in 4 ~1 of water, and loaded into a single lane of the polyacrylamide gel. The gel was dried and exposed to an X-ray film at room temperature. The molecular size marker used was the 123-bp ladder from BRL. RESULTS
Using a single downstream Alu-specific primer, we tested the feasibility of alumorph detection (cf Fig. 1) by PCR amplification of genomic DNAs of five unrelated individuals of different ethnic backgrounds. The results using undigested genomic DNAs or genomic DNAs digested with the B&XI restriction endonuclease are shown in Fig. 2. A characteristic pattern is observed in all individuals; however, certain variable fragments distinguish the five individuals tested. As expected, the amplification pattern generated from BstXI-digested DNA samples (Fig. 2B) is less complex than that for undigested DNA, due to the disappearance of several fragments in which a BstXI site is presumably present. In BstXI digests, more than 50 bands per individual sample may be distinguished, at least 15 of which appear to be variable. Figure 3 compares PCR-amplified DNA fragments from BstXI digests of DNA samples isolated from 10 individuals of a single pedigree. Here again, distinct amplification patterns for different individuals are observed. Most importantly, the pattern of fragments observed in offspring is a composite of those found in their parents. This is consistent with Mendelian segregation of alleles and demonstrates that alumorphs can be used as genetic markers. DISCUSSION
It has been estimated that 200 to 400 informative markers, equally distributed throughout the human
333
ALUMORPHS
genome, would permit the localization of any genomic locus (Bishop and Skolnick, 1983). We have shown that 10 to 15 polymorphic sites can be revealed in a single PCR assay. Distinct polymorphic sites could be detected with various combinations of Ah primers and enzymatic restiction digests. Therefore, less than 100 assays would be required to cover the entire genome. Furthermore, despite their preferential hybridization to chromosomal R-bands, Ah repeats are ubiquitous in human DNA (Moyzis et al., 1988, Korenberg and Rykowski, 1988). Consequently, a quasi-random distribution of alumorphs in the genome may be expected, a feature that is important for mapping. This is in contrast to the distribution of hypervariable minisatellite or VNTR markers which tend to cluster in the proterminal regions of human autosomes (Royle et al., 1988, Armour et al., 1989). The fact that alumorphs are dominant markers, unless quantitative analysis is applied, renders them less informative than classical RFLP markers. However, this can be compensated for by increasing the overall density of markers facilitated by multiple polymorphisms detected in a single experiment. This approach may be compared to amplified sequence polymorphisms, ASPS (Skolnick and Wallace, 1988), except that, for alumorphs, prior knowledge of DNA sequence of each amplified polymorphic locus is not required. For physical mapping, alumorphic fragments of appropriate length can be excised from the A 12345
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The PCR amplification pattern of B&XI-digested of 10 individuals from a single family (experimental were as in Fig. 2, except that electrophoresis was carried polyacrylamide gel). In the family tree the symbol for overlies the corresponding amplified DNA sample. are indicated on the left side of the autoradiogram.
DNA conout each Size
gel, reamplified by PCR, and used to generate singlecopy probes for in situ or chromosomal panel hybridizations, as recent experiments suggested (Nelson et at., 1989). Such sequences could become an important source of STSs (sequence-tagged sites), recently recommended as reference points for the physical mapping of the genome (Olson et al., 1989). In addition to mapping studies, alumorphs may find use in genotyping for forensic investigations and population genetics as well as in phylogenetic studies of primates. Finally, this approach may be generalized to any genome where interspersed repetitive sequences are found. ACKNOWLEDGMENTS
FIG. 2. Amplification patterns of five unrelated individuals were obtained by PCR amplification of genomic DNA samples with a single Ah-specific oligonucleotide primer. In (A), nondigested DNAs isolated fmm peripheral blood have been used, whereas those in (B) were digested with the B&XI restriction endonuclease prior to PCR amplication. Positions of the molecular weight markers are indicated to the left of the autoradiogram.
We are indebted to Dr. Grant Mitchell for his comments and discussions and to Drs. Robert Cedergren and Pierre Chartrand for their critical comments on the manuscript. This work was supported by a grant from the Cancer Research Society Inc. D.S. is a recipient of a studentship from the Medical Research Council of Canada, J-M.D. is supported by the Fondation de l’H6pital &e-Justine (HSJ) and the Service de Genetique M&&ale of HSJ, L.R.S is supported
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by the Muscular Dystrophy Association of Canada, and D.L. has a scholarship of the Fonds de Recherche en Sante du Quebec. REFERENCES 1. ARMOUR, J. A. L., WONG, Z., WILSON, V., ROYLE, N. J., AND JEFFREYS, A. J. (1989). Sequences flanking the repeat arrays of human minisatellites: Association with tandem and dispersed repeat elements. Nucleic Acids Res. 17: 4925-4935. 2. BISHOP, D. T., AND SKOLNICK, M. H. (1983). Genetic markers and linkage analysis. Banbury Rep. 14: 251-259. 3. BOTSTEIN, D., WHITE, R. L., SKOLNICK, M. H., AND DAVIS, R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Amer. J. Hum. Genet. 32: 314-331. 4. BRITTEN, R. J., BARON, W. F., STOUT, D. B., AND DAVIDSON, E. H. (1988). Sources and evolution of human Alu repeated sequences. Proc. Natl. Acad. Sci. USA 86: 4770-4774. 6. DEININGER, P. L., JOLLY, D. L., RUBIN, C. M., FRIEDMAN, T., AND SCHMID, C. W. J. (1981). Base sequence studies of 300 nucleotide renatured repetitive human DNA clones. J. Mol. Biol. 151: 17-33. 6. JEFFREYS, A. J., WILSON, V., NEUMANN, R., AND KEYTE, J. (1988). Amplification of human minisatellites by the polymerase chain reaction towards DNA fingerprinting of single cells. Nucleic Acids Res. 16: 10,953-10,971. 7. JURKA, J., AND SMITH, T. (1988). A fundamental division in the Alu family of repeated sequences. Proc. Natl. Acad. Sci. USA
86: 4775-4778.
8. KORENBERG, J. R., AND RYKOWSKI, M. C. (1988). Human genome organization: Alu, Line and the molecular structure of metaphase chromosome bands. Cell 53: 391-400. 9. LABUDA, D., AND STRIKER, G. (1989). Sequence conservation in Alu evolution. Nucleic Acids Res. 17: 2477-2491. 10. LIIT, M., AND LUTY, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Amer. J. Hum. Genet. 44: 397401. 11. MOYZIS, R. K., TORNEY, D. C., MEYNE, J., BUCKINGHAM, J. M., WV, J-R., BURKS, C., SIROTKIN, K. M., AND GOAD, w. B. (1989). The distribution of interspersed repetitive DNA sequences in the human genome. Genomics 4: 273-289.
ET AL. 12. 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 236: 1616-1622. 13. NELSON, D. L., LEDBETTER, S. A., CORBO, L., VICTORIA, M. F., RAMIREZ-SOLIS, R., WEBSTER, T. D., LEDBE~R, D. H., AND CASKEY, C. J. (1989). Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA 86: 6686-6690. 14. OLSON, M., HODD, L., CANTOR, C., AND BOTSTEIN, D. (1989). A common language for physical mapping of the human genome. Science 245: 1434-1435. 15. ROBERTS, R. G., COLE, C. G., HART, D. A., BOBROW, M., AND BENTLEY, D. R. (1989a). Rapid carrier and prenatal diagnosis of Duchenne and Becker muscular dystrophy. Nucleic Acids Res. 17: 811. 16. ROBERTS, R. G., MONTANDON, A. J., BOBROW, M., AND BENTLEY, D. R. (1989b). Detection of novel genetic markers by mismatch analysis. Nucleic Acids Res. 17: 5964-5971. 17. ROYLE, N. J., CLARKSON, R. E., WONG, Z., AND JEFFREYS, A. J. (1988). Clustering of hypervariable minisatellites in the proterminal regions of human autosomes. Genomics 3: 352360. 18. SAIKI, R. D., SCHARF,S. J., FALOONA, F., MULLIS, K. B., HORN, G. T., ERLICH, H. A., AND ARNHEIM, H. (1985). Enzymatic amplification of &globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 13501354. 19. SINNETT, D., LAVERGNE, L., MELANCON, S. B., DALLAIRE, L., POTIER, M., AND LABUDA, D. (1988). Lesch-Nyhan syndrome: Molecular investigation of three French Canadian families using a hypoxanthine-guanine phosphoribosyltransferase cDNA probe. Hum. Genet. 81: 4-8. 20. SKOLNICK, M. H., AND WALLACE, T. B. (1988). Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPS). Genomics 2: 273-279. 21. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. 22. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Amer. J. Hum. Genet. 44: 366-396. 23. WHITE, R., AND LALOUEL, J.-M. (1988). Sets of linked genetic markers for human chromosomes. Annu. Rev. Genet. 22: 259279.