Genetic marker technology AI Edwards and C. Thomas Caskey Institute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas, USA Advances in our understanding of polymorphisms found in eukaryotic genomes and improved methods for studying genetic markers should facilitate genetic linkage mapping and other applications. Progress within the past year includes characterization of the types, frequencies, and properties of tandemly repeated sequences, methods for obtaining the DNA sequence flanking genetic markers for use in the polymerase chain reaction, and new detection systems featuring automation and multiplexing. Current Opinion in Biotechnology 1991, 2:818-822
Introduction Polymorphic genetic markers are the foundation of many strategies used to study individuals and populations. Progress in genetic linkage mapping, DNA diagnostics, and population genetics is closely tied to the development of methods for studying genetic markers and the identification of new types of polymorphisms. The invention of the polymerase chain reaction (PCR) in 1985 [1] revolutionized genetic marker technology by providing rapid and precise access to genomic polymorphisms on the scale of a few base pairs to a few kilobases. Of particular importance to genetic marker technology, the PCR enabled 1-5 bp short tandem repeats (STRs), present in many eukaryotic genomes to be used as genetic markers. This was a significant advance for linkage mapping and other genetic studies. The past year has seen an improved understanding of the properties of different classes of STRs, their frequencies in the human genome, and methods for studying them [2",3,4,5"]. Strategies for studying different classes of A/u polymorphisms [3,6.,7",8,9 "] and Qt-satellite variation [10] have been described. Single base pair potymorphisms have been rendered more efficient with the application of the PCR and new detection strategies [11.,12,13]. The incorporation of these advances into applications has occurred simultaneously [5,11",14-16].
Single nucleotide substitutions Polymorphisms of a single or a few nucleotide positions occur approximately once in every 1000bp (Fig. 1). Although these genetic markers are the most common, they have the disadvantage of being two-allele systems with low heterozygote frequencies. Single nucleotide poly-
morphisms were originally detected as restriction fragment length polymorphisms (RFLPs) by Southern hybridization [ 17]. More recently, they have been detected by chemical mismatch cleavage [18] and denaturation gradient gel shifts [19]. The use of PCR facilitated the identification and detection of these potymorphisms by obviating the need for Southern hybridization [20]. In each case, PCR makes use of the DNA sequence flanking each of the polymorphic sites. The application of fluorescent and colorimetric detection systems to amplified single nucleotide polymorphisms creates the potential for complete automation. Primer-guided nucleotide incorporation assays [12], oligonucleotide ligation assays (OLA) [11"], and fluorescentIy Labelled priming oligonucleotides [13] have been applied to these polymorphisms. One method used employs two levels of specificity(first, amplification of the target by PCR and, second, OLA) with a colorimetric ELISA for digoxigenin [11.]. The results obtained are simple to interpret, and with automation may prove valuable for such applications as DNA diagnostics, personal identification, and genetic linkage when high throughput is desired.
Tandemly repeated sequences Variable number of tandem repeats and satellite polymorphisms Variable number of tandem repeat (VNTR) polymorphisms often have heterozygosities in the range of 60% to 99% and have therefore been useful for application to disease gene localization in small kindreds and DNA typing (e.g. personal identification) since their introduction [21,22]. Traditionally assayed by Southem blot, the VNTR loci, can now be studied with PCR [23"',24], however, their long repeat motif (for example 15bp) and total
Abbreviations OLA---oligonucleotide ligation assays;PCR--polymerasechain reaction; iFLP--restriction fragment length polymorphism; $SCP--single stranded conformation polymorphism; STR'v--~shorttandem repeats;VN111--variablenumber of tandem repeats. 818
(~) Current Biology Ud ISSN 0958-1669
Genetic marker technology Edwards and Caskey
S0 kb tw
I
n'~,~
~ x , . = = . . -- = ~ _ = g , ~ q . = . . ~
•
I
Point m u t a t i o n
("-1/1000 bp) Line I ("-1/30 kb)
[~Alu
repeat
['7]
[]
a~,
u
~r-,~n~
--',ta- - ~
STR
(-,-1/10 kb) Done hower repeat
(-,-1/20 kb)
(-,-1/5 kb)
Fig. 1. Schematic diagram of the approximate frequency and distribution of polymorphic genetic markers on an idealized segment of genomic DNA. Short tandem repeats are derivated by SIR.
length (for example a few kb) render uniform amplification of all alleles more difficult than withthe more recently described 2-5 bp tandem repeats. Furthermore, the total number of VNTRs in the genome is thought to be orders of magnitude lower than that of STRs (compare [25] and [5"]) and the difficulty of precise ~ e l e identification [26] has tempered enthusiasm for their further development and use in genetic mapping and DNA diagnostics. Despite this, VNTRs ~ continue to be used for many applications. For example, the VN'rR locus D I S 8 w a s observed to have > 1070 alleles when the internal core repeats of amplified alleles are scored for the presence or absence of a variable restriction site [23"']. This is an example of a compound polymorphism, which is also seen with STRs (A Edwards and CT Caskey, unpublished results). Satellite DNA found at the primary constriction of chromosomes is known to be highly repetitive. The variability of this satellite DNA has been studied with pulse field gel electrophoresis (PFGE) and long range restriction mapping by using restriction enzymes that rarely cut the satellite DNA, but frequently cut unique DNA. Using these techniques, the human X chromosome cx-sateUite array was shown to vary between 1380 kb and 3730 kb and Mendelian inheritance was observed [10]. These pulse field gel electrophoresis polymorphisms should be useful for linkage to nearby disease genes.
Short tandem repeat polymorphisms Comparison of orthologous DNA sequences and early mutation studies identified tandemly repeated sequences only a few base pairs in length (1-5bp) as regions with higher mutation rates than adjacent single copy sequences [27,28]. It was not until the application of PCR, however, that these markers were successfully studied, and the hypervariability of STRs appreciated [29-33]. The past three years have seen characterization of a large number of dimeric (micro-sateUite), trimeric, and
tetrameric tandem repeats. The relative ease with which they may be amplified, the potential for sequence-based allele identification, uniform dislike across the genome, and their high heterozygosity make them particularly attractive markers. STRs appear to be the third most common class of polymorphic marker found in the human genome, being approximately one half as abundant as A/u repeats. The most frequent STRs a r e dimeric [AC] sequences [34] that are found every 20-30 kb in genomic DNA. Hybridization of five oligonudeotides complementary to specific STRs (e.g., [AGAT]75and [AAT]10) to genomic DNA suggested that trimeric and tetrameric STRs are found once every 300-500 kb [5"] in genomic DNA. As there are 44 unique trirnefic and tetrarneric sequences, the total frequency of trimeric and tetrameric STRs could be as high as one STR per 9 kb; that is, about 200 000 polymorphic STR markers in the human genome. The latter calculation makes the reasonable assumption that 50% or more of the STRs will be polyrnorphic [2",5"]. For unknown reasons, one STR locus may be hyper~-ariable, whereas another locus of the same core sequence and number of repeats may be monomorphic [5"]. It has been shown for dimeric STP,s that loci with greater numbers of repeats (e.g. [AC]20versus [AC]10) are more likely to be polymorphic and have higher heterozygote frequencies [2.]. The data available for trimeric and tetrameric STRs are consistent with the [AC] data [5"]. Incorporating this concept into strategies for identifying randomly distributed polymorphic STRs for genetic mapping and other applications should increase the fracdon of potymorphic STRs to total STRs characterized to more than 75%. There is no evidence that the particular core sequence influences either the probability of a STR being polymorphic or heterozygote frequencies at STR loci are influenced by the particular core sequence. The high heterozygotic and genomic frequencies of STRs has enabled linkage to disease loci in small families [15] and
819
820 Mammaliangene studies the use of an anonymous STR to achieve linkage to the facioscapulohumeral dystrophy locus [16]. Although the hypervafiability and high genomic frequency of [AC]n repeats make them powerful genetic markers, one problem has been interpreting the banding profiles. Generation of repeat length insertion and deletion (presumed slippage) products often creates a 2 bp ladder effect after gel electrophoresis. Additionally, the terminal transferase activity of the thermostable polymerase [35] may convert one or both strands of the 2 b p ladder into a 1 bp ladder if the reaction does not proceed to completion. Consequently, interpretation of the allele profiles may be difficult with particular loci, especially when non-familial samples are being typed. Although the terminal transferase activity is seen with tfimeric and tetrameric STRs, generation of presumed slippage products is much less of a problem [5"], and the longer core motif sequence facilitates allele identification. The amplification fidelity of trimeric and tetrameric STRs has enabled the development of a STR-based DNA typing assay featuring multiplex PCR [36], internal standards and fluorescent detection of alleles [5"]. Most applications of STRs would be facilitated by the availability of methods both to ascertain whether STRs are polyrnorphic apm'om'and to rapidly obtain flanking DNA sequences from which PCR primers could be designed. A reasonable solution to the first problem is selection for trimeric and tetrameric STRs with more than seven repeats. The second problem remains to be solved decisively but a PCR strategy [37 o] for amplifying the ends of YAC inserts using a double stranded oligonucleotide anchor containing an internal region of non-complementarity has been applied to STRs [5"]. By amplifying the sequence between the STR and the non-complementary region of the anchor with primers complementary to the STR and the anchor, DNA segments flanking several [AGAT]n STRs were amplified [5"]. This method has been applied to other STR sequences (I-I~ Hammond, A Edwards, CT Caskey, unpublished results). As experience is accumulated, the utility of the method relative to random strategies [2.] will be determined. Polymorphic trimeric STRs have been found within the coding regions of genes that include the human androgen receptor gene [5"], the Drosophila Notch gene and related genes [32,38], and the gene for the human fragile X locus [39]. Variable tandem reiteration of much longer sequences has also been reported for apolipoprotein A [40] and mucin-type glycoprotein genes [41]. Abnormally high numbers of core repeats (CAG, glutamine) in the hypervafiable trimeric STR within the human androgen receptor genes have been associated with spinal and bulbar muscular dystrophy [14] and other androgen receptor defects [42]. The polymorphic STR within the candidate fragile X locus (CGG, arginine) is involved in the mutations accounting for this disease (Ying Hui Fu et aL, and CT Caskey unpublished results, [39]). These results suggest a strategy' for the identification of disease genes.
Alu polymorphisms The middle repetitive, interspersed Alu repeat occurs approximately 500 000 times in the human genome. It is the most common repetitive sequence in the human genome, occurring on average every 5 kb in genomic DNA. By coupling amplification of Alu repeats in PCRs using primers complementary to unique flanking DNA segments with the recently described technique of singlestrand conformation polymorphism (SSCP) [9",43], approximately 50% of A/u repeats studied were found to be polymorphic [9"]. Heterozygote frequencies ranged from 0.06-0.63 (average 0.38) [9"]. The SSCP method detects polymorphisms by conformational differences observed in single stranded DNA sequences after electrophoresis under non-denaturing conditions, and therefore does not require knowledge of the nudeotide alterations [43]. The use of the SSCP method does, however, require knowledge of the flanking sequence for use with PCIL Most of the A/u polymorphisms detected appear to be single nucleotide substitutions, deletions, or insertions [9o] and thus these polymorphisms may also be incorporated into the automated methods described above. A/u elements are thought to be propagated throughout the genome via retroposition [44]. During this process a poly(A) tail approximately 40 nucleotides long is added to the A/u transcript before insertion into the genome [45]. A cursory review of Alu repeats in GenBank reveals, however, that a significant fraction (6%) of the poly(A) tails become tandemly repeated [(A)nGn] and [(A)nC n] sequences [3]. A fraction of these STRs have been found to be polymorphic, and are useful as genetic markers [3]. in addition to the utility of these sequences as genetic markers, the results imply a mechanism by which other interspersed non-A/u associated STRs may have evolved. A study of the divergence of A/u sequences in genetic databases has led to the subdivision of Alu elements into different classes, thought to be of different genetic ages [46]. The human-specific class is thought to have been transposed in recent human evolution. An oligonucleotide designed to distinguish this class of Alu from approximately 500 000 older repeats enabled the identification of recently transposed sequences in the human genome [8,45,47]. Some of these are insertion polymorphisms for humans, and may provide markers for the study of human evolution.
Arbitrarily primed PCR in the technique of abitrarily primed PCR the amplification is performed with a single arbitrarily chosen primer [48-50]. The amplification products constitute a fingerprint that can be used to distinguish between strains of an organism. The advantage of this technique is that DNA sequence information is not required and it can therefore be applied to any genomic DNA sample for strain identification or other studies.
Genetic marker technology Edwards and Caskey 821 Conclusions
A u-anspositional~" ac~'e A/u subfamily and dimorphismof represenm~ s of this family are described.
Marked advances have been made in our understanding and use of polymorphic genetic markers since the invention of the PCR. Continued application of semi-automated colorimetric and fluorescent detection schemes and multiplexing (the amplification of several markers simultaneously) to STRs should provide the power and Offciency needed for the genetic mapping of large genomes. STRs are also suitable for correlating genetic and physical maps. Because the new mapping approaches are not derivatives of the original two-allele or VNTR polymorphisms, and the development of the new PCR-based markers is proceeding rapidly, it would appear to be more efficient to develop a new set of genetic markers than to continue retrofitting of older, less useful RFLPs. The new polymorphic genetic markers discussed above should provide a rapid method of establishing a genetic map of man and easily used methods for increased the understanding of our genome, its evolution, and gene loC~-liT~qtions.The new PCR-based methods will predictably have wide application in human genetics for an extended period of time.
7.
BAT'ZERMA, DEINI~GER Pt. A Human-Specific Subfamily of
•
Alu Sequences. Genom/cs1991, 9:481-487.
Demonstration of a pob'morphism within the human specific A/u sub-
famib'. 8.
BATT.ER MA, GUDI VA, MENA JC, FOLTZ DW, HERRERA RJ, DEI~2S~GERPL: Amplification Dynamics of Human-Specific (HS) alu Family Members. Nucleic Acids Res 1991, 19:3619-3623.
9. OmTAM, SEKIYAT, HAYASmK: DNA Sequence Polymorphisms • in Alu Repeats. Genom~.s 1990, 8:271-278. A comprehensive study of the types of polymorphisms detected by the sin#e-strand conformation poMnorphismmethod. 10.
MAHTA~aMM, WnIARD HF: Puised.field C~I Analysis of orsatellite D N A at the Human X Chromosome Centromere: High-frequency Polymorphisms and Array Size Estimate. Go nom/cs 1990, 7:607-613.
11.
NICKERSON DA, KAISER P~ LAPPIN S, STEWARTJ, HOOD L,
•
LANDEGRENU: Automated DNA Diagnostics using an ELISA-
based Oligonucleotide Ligation Assay. Proc Nail Acad $ci USA 1990, 87:8923-8927. Description of a potentially automated non-isotopic system for DNA diagnostics using PCR to amplify DNA target segments. 12.
SYVANENA-C, AM.TO-SETALAK, HARJUL, Korqru~ K, SODERLUND H: A Primer-guided Nucleotide incorporation Assay in the Genotyping of Apolipoprotein E. Genom~cs 1990, 8:684-692.
Acknowledgements
13.
A Edwards is a Josephine and Edward Hudson Scholar and Medical S~ntist Training FeUow. CT Caskey is a Howard Hughes Medical Institute Investigator.
CHEHABFF, KAN YW: Detection of Specific DNA Sequences by Fluorescence Amplification: a Color C o m p l e m e n t a t i o n Assay. Prc~ Nail Acad Sci USA 1989, 86.9178--9182.
14.
LA SPADAAR, WILSONEM, lamAia~ DB, HARDINGAE, FISCHBECK KH: Androgen Receptor Gene Mutations in X-linked Spinal a n d Bulbar Muscular Atrophy. Nature 1991, 352:77-79.
15.
HUA~G TH-M, ~ C t K JF, EDWARDS A, P~-nG~EW L H E m ~ CA, HAMMONDHA, CASKEYCT, ZOGHBtHY, I2tmETTER DH: Linkage of the Gene for an X-linked Mental Retardation Disorder to a Hyt~rvariable (AGAT)n Repeat Motif within the Human HPRT Locus (Xq26). Am J Hum Genet 1991, in press.
16.
W[JME~,A C, FRAmS RR, BROU~R OF, MOERER P, WEBER JL, PADBERG GW: Location of Fascioscapulohumeral Muscular Dystrophy Gene o n Chromosome 4. Lancet 1990, 336:651--653.
17.
MYERSRM, LUMELSKYN, LERMANIS, MANIA'mT: Detection of Single Base Substitutions in Total Geneomic DNA. Nature 1985, 313:495--498.
18.
KAN YW, DOZY AM: Pol)maorphism of DNA Sequence Ad. jacent to Human Beta Globin Structural Gene: Relationship to Sickle Mutation. Proc Naa m a d S a USA 1978, 75:5631-5635.
19.
COTIONRGH, RODRIGUESNR, CAMPBELLRE): Reactivity of Cytosine and Thymine in Single-base-pair Mismatches with I-b/droxylamine and Osmium Tcrtroxide and its Application to the Study of Mutations. Proc Nail Acad Sci USA 1988, 85:4397-4401.
20.
SABORK, SCHARFS, FAIDONA F, MULLISIO3, HORN GT, ERLICH HA, /m.'~tzL~ N: Enzymatic Amplification of 13-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cel] Anemia. Science 1985, 230:1350-1354.
21.
NA~MURA Y, LEPPERT M, O'CONNEL P, WOLFF IL HOLM T, CULVERM, M.M~rI~ C, FLrJIMOTOE, HOFF M, KUMLINE, WHn'E l~ Variable Number of Tandem Repeat (VNTR) Markers for Human Gene Mapping. Science 1987, 235:1616-1622.
22.
JEFFREYSAJ, WmSONV, THEIN St- Hypervariable 'Minisatellitc' Regions in Human DNA. Nature 1985, 314.67-73.
References and recommended reading Papers of special interest, published within the annual period of review,
have been hightishtedas: • •.
of interest of outstanding interest
1.
MULUSK, FALOONAF, SCHARF S, SAIKI R, HORN G, EmaCH H: Specific Enzymatic Amplification of DNA in vttr~ the Poiymerase Chain.Reaction. Co/d Spring Harbor Syrup Quant B/o/1986, 51:263-273.
2. ~he
WEBERJt. Informativeness of Human (dCMA)n-(dG-dT)n Polymorphisms. Genom/ca 1990, 7:524-530. author discusses factors that predict whether, or not, a given (AC) repeat will be polymoprhic and the associated heterozygote frequencies. 3.
ECONOMOUEP, BERGEN AW, WARREN AC, ANTONARAKISSE: The Polydeoxyadenylate Tract of Alu Repetitive Elements is Polymorphic in the Human Genome. Proc Natl Acad Set USA 1990, 87:2951-2954.
4.
EPSTEINN, NAHORO, SIXVERJ: The 3' Ends of Alu Repeats are Highly Polymorphic. N u d e ~ Ac/ds Res 1990, 18;4634--4634.
5. •
EDWARDSA, CrerrELLOA, HAMMONDHA, CASKEYCT: DNA TypLag and Genetic Mapping with Trimeric and T e m e r i c Tandem Repeats. Am J Hum Genet 1991, 49:746-757. The genomic frequency and other properties of trimeric and tetrameric tandem repeats in humans is discussed. A method for obtaining DNA sequence flanking STRs and a fluorescent multiplex DNA typing are presented. 6. •
MATERAAG, HE~'~,'N U, SCHMZD CW: A Transpositionally and Trancriptionally Competent Alu Subfamily. Mol Cell Biol 1990, 10:5424-5432.
822
Mammalian g e n e studies JEFFRE~AJ, NEUMANNR, WI~ON V: Repeat Unit Sequence Variation in MinisateUites: a Novel Source of DNA Polymorphism for Studying Variation and Mutation by Single Molecule Analysis. Cell 1990, 60:473-485. Descriptjo' n of an elegant assay for DNA typing and study of the evolution of VN'IP,s. Jeffre~ et al provide direct estimates for the mutation rate of a VNTR locus and describe the structure of the mutated aUeles.
Description of an anchored-PCR method ~ith significant ack~ntages over other methods. 38.
WHARTONK, YEDVOBN1CKB, FtNNEtrrYV, ARTAVANIS-TSAKO.',~ASS: opa: A Novel Family of Tr-an~ril~d Repeats Shared by the Notch Locus and Other Developmentally Regulated Loci in D. melanogaster. Cell 1985, 40:55-62.
24.
JEFFREY5AJ, WILSONV, NEUMANNK KEYIXJ: Amplification of Human Mini-satellites by the Polymerasc Chain Reaction: Towards DNA Fingerprinting of Single Cells. Nucleic Acids Res 1988, 16:10953-10971.
39.
25.
ARMOURJA, POVEYS, JEREMIAHS, JEFFREYSAJ: Systemic Cloning of Human Mini-Satellites from Ordered Array Charomid Libraries. Genom/cs 1990, 8:501-512.
VERk~RKAJMH, PmRETn M, SUTO.WFEJS, FU Y-H, KUHL DPA, P1ZZUTI A, RELNERO, RICHARDSS, VICTORIAME, ZHANG F, ET at.: Identification of a Gene (FMR-1) containing a CGG Repeat Coincident with a Breakpoint Cluster Region Exhibiting Length Variation in Fragile X Syndrome. Cell 1991, 65:905-914.
40.
KOSCH~SKYM~ BELSIEGELU, HENNE-BRUNSD, EATONDL, LAWN RM: Apolipoprotein A Size Heterogeneity is Related to Variable Number of Repeat Sequences in its mRNA. B ~ e r n ~try 1990, 29:640-644.
41.
SWALLOWDM, GENDLER S, GRtrrnvLS B, CO~XY G, TAYLORPAPADIMrrRIouJ, BRAMWELLME: The Human Tumour-associated Epithelial Mucins are Coded by an Expressed Hypcr,,-ariable Gene Locus PUM. Nature 1987, 328:82-84.
42.
MCPHAULMJ, MARCELU M, Tnazv WD, GRtFF~ JE, ISlImOGmaERREZ RF, WILSONJD: Molecular Basis of Androgen Resistance in a Family with a Qualitative Abnormality of the Androgen Receptor and Responsive to High-dose Androgen Therapy. J Clin Invest 1991, 87:1413-1421.
43.
OmTA M, SUZUKl Y, SEKIYA T, tIAYASm K: Rapid and Sensitive Detection of Point Mutations and DNA Polymorphisms using the Polymerasc Chain Reaction. Genomica 1990, 5:874-879.
44.
ROGERSJ: Retroposons Defined. Nature 1983, 301:460-460.
45.
BATZERMA, KILROYGE, ~CHARD PE, SHAtKHTH, DESSELLETD, HOPPENS Cl~ DEL~q2qGERPL: Stl~cture and Variability of Recently Inserted Alu Family Members. Nucleic Acids Res 1991, 18:6793-6798.
46.
SLAGELV, FLEMLNGTON E, TRA~A-DORGE V) BRAI~HAW H, DE1NLNGERPI2 Clustering and Subfamily Relationships of the Alu Family in the Human Genome. Mo/ B/o/ EL~/ 1987, 4:19-29.
47.
MArERAAG, HEILMANN U, HINTZ MF, SCHMID CW: Recently Tt-ansposed Alu Repeats Result from Multiple Source Genes. Nucleic Acids Res 1991, 18:6019--6023.
48.
WIILIAMSJGK, KUBELIK AR, LWAK KJ, RAFALSKIJA, "I~GEY SV; DNA Polymorphisms Amplified by Arbiwary Primers are Useful as Genetic Markers.. Nucleic Acids Res 1990, 18:6531-6535.
49.
WELSHJ, McCLELUe~ M: Fingerprinting Genomes Using PCR with Arbitrary Primers. Nucleic Acids Res 1991, 18:7213-7218.
50.
WELSHJ, P~XRSEN C, MCCLEtL~','DM: Polymorphisms Generated by Arbitrarily Primed PCR in the Mouse: Application to Strain Identification and Genetic Mapping. Nucleic Acids Res 1991, 19:303-306.
23. **
26.
DEvI~ B, R1SCH N, ROEDER K: No Excess of Homozygosity at Loci Used for DNA Fingerprinting. Sc/ence 1990, 249:1416-1420.
27
SAVATIERP, "I"~d3UCHETG, FAURE C, CHEBLOUNEY) GODY M, VERDIER G, NIGON VM: Evolution of the Primate ~-Globin Gene Region: High Rate of Variation in CpG Di.nucleotides and in Short Repeated Sequences between Man and Chimpanzee. J Mo/B/cd 1985, 182:21-29.
28,
SEMENZAGL, MAUADI P, SURREY S, DELGROSSO K, PONCZ M, SCHWAR'IZ E: Detection of a Novel DNA Polymorphism in the 13-globin Gene Cluster. J Biol O~em 1984, 259:6045-6048.
29.
WEBERJL, MAY PE: Abundant Class of Human DNA Polymorphisms which can be Typed Using the Polymerasc Chain Reaction. Am J Hum Genet 1989, 44:388-396.
30.
Lrrr M, U.rry JA: A Hypervarlablc Microsatellite revealed by in vitro Amplification of a Di-nucleotidc Repeat Within the Cardiac Muscle Actirt Gene. Am J Hum Genet 1989, 44 -.397---401.
31.
Dm0A "I'P, MUKAI S, PETERSON R, RAPAPORTJM, WALTON D, YANDELLDW: Parental Origin of Mutations of the Retinoblastoma Gene. Nature 1989, 339:556-558.
32.
TALrm D: Hypcrvarlabilityof Simple Sequences as a General Source for Polymorphic D N A Markers. Nu~eic Acids Res 1989, 17:6463-6471.
33.
EDWARDS A, GraBS RA, NGUYEN PN, ANSORGE W, CASKEY CT: Automated D N A Sequencing Methods for Dctcction and Analysis of Mutations: Applications to the Lesch-Nyhan Syndrome. Tram Assoc Am Physiciam 1989, CII:185--194.
~P~.
STAIMNGSR]., FORD AF, NELSON D, TORNEY DC, HOLDEBRA.ND CE, MoYzls RK: Evolution and Distribution of (GT)n RepetRive Sequen~:es in Mammalian Genomes. Genom/ca 1991, 10:807-815.
35.
CLARKJM: Novel Non.tcmplated Nucleodde Addition Reactions Catalyzed by Prokaryotic and Eukaryotic DNA Polymer. Nuc/e~ Adds Res 1988, 16:9677-9686.
36.
CHAMBERtA~JS, GraBS RA, RANTERJE, NGUYENP-N, CASKEYCT: Deletion Screening of the Duchcnne Muscular Dystrophy Locus via Multiplex DNA Amplification. Nuc/e/c Adds Re$ 1988, 16:11141-11156.
37.
Rn.EYJ, BUTLERR, OGILVIE D, FINNmAR R, JENNER D, POWELL
*
S, ANAND R, SMITHJC, MARKHAMAF: A Novel Rapid Method
for the Isolation of Terminal Sequences from Yeast Artitidal Chromosome (YAC) Clones. Nucleic Acids Res 1990, 18:2887-2890.
A Edwards and CT Caskey, Institute for Molecular Genetics, and Howard Hughes Medical Institute, Baytor College of Medidne T809, 1 Baylor Plaza Houston, Texas 77030, USA.
Introduction Polymorphic genetic markers are the foundation of many strategies used to study individuals and populations. Progress in genetic linkage mapping, DNA diagnostics, and population genetics is closely tied to the development of methods for studying genetic markers and the identification of new types of polymorphisms. The invention of the polymerase chain reaction (PCR) in 1985 [1] revolutionized genetic marker technology by providing rapid and precise access to genomic polymorphisms on the scale of a few base pairs to a few kilobases. Of particular importance to genetic marker technology, the PCR enabled 1-5 bp short tandem repeats (STRs), present in many eukaryotic genomes to be used as genetic markers. This was a significant advance for linkage mapping and other genetic studies. The past year has seen an improved understanding of the properties of different classes of STRs, their frequencies in the human genome, and methods for studying them [2",3,4,5"]. Strategies for studying different classes of A/u polymorphisms [3,6.,7",8,9 "] and Qt-satellite variation [10] have been described. Single base pair potymorphisms have been rendered more efficient with the application of the PCR and new detection strategies [11.,12,13]. The incorporation of these advances into applications has occurred simultaneously [5,11",14-16].
Single nucleotide substitutions Polymorphisms of a single or a few nucleotide positions occur approximately once in every 1000bp (Fig. 1). Although these genetic markers are the most common, they have the disadvantage of being two-allele systems with low heterozygote frequencies. Single nucleotide poly-
morphisms were originally detected as restriction fragment length polymorphisms (RFLPs) by Southern hybridization [ 17]. More recently, they have been detected by chemical mismatch cleavage [18] and denaturation gradient gel shifts [19]. The use of PCR facilitated the identification and detection of these potymorphisms by obviating the need for Southern hybridization [20]. In each case, PCR makes use of the DNA sequence flanking each of the polymorphic sites. The application of fluorescent and colorimetric detection systems to amplified single nucleotide polymorphisms creates the potential for complete automation. Primer-guided nucleotide incorporation assays [12], oligonucleotide ligation assays (OLA) [11"], and fluorescentIy Labelled priming oligonucleotides [13] have been applied to these polymorphisms. One method used employs two levels of specificity(first, amplification of the target by PCR and, second, OLA) with a colorimetric ELISA for digoxigenin [11.]. The results obtained are simple to interpret, and with automation may prove valuable for such applications as DNA diagnostics, personal identification, and genetic linkage when high throughput is desired.
Tandemly repeated sequences Variable number of tandem repeats and satellite polymorphisms Variable number of tandem repeat (VNTR) polymorphisms often have heterozygosities in the range of 60% to 99% and have therefore been useful for application to disease gene localization in small kindreds and DNA typing (e.g. personal identification) since their introduction [21,22]. Traditionally assayed by Southem blot, the VNTR loci, can now be studied with PCR [23"',24], however, their long repeat motif (for example 15bp) and total
Abbreviations OLA---oligonucleotide ligation assays;PCR--polymerasechain reaction; iFLP--restriction fragment length polymorphism; $SCP--single stranded conformation polymorphism; STR'v--~shorttandem repeats;VN111--variablenumber of tandem repeats. 818
(~) Current Biology Ud ISSN 0958-1669
Genetic marker technology Edwards and Caskey
S0 kb tw
I
n'~,~
~ x , . = = . . -- = ~ _ = g , ~ q . = . . ~
•
I
Point m u t a t i o n
("-1/1000 bp) Line I ("-1/30 kb)
[~Alu
repeat
['7]
[]
a~,
u
~r-,~n~
--',ta- - ~
STR
(-,-1/10 kb) Done hower repeat
(-,-1/20 kb)
(-,-1/5 kb)
Fig. 1. Schematic diagram of the approximate frequency and distribution of polymorphic genetic markers on an idealized segment of genomic DNA. Short tandem repeats are derivated by SIR.
length (for example a few kb) render uniform amplification of all alleles more difficult than withthe more recently described 2-5 bp tandem repeats. Furthermore, the total number of VNTRs in the genome is thought to be orders of magnitude lower than that of STRs (compare [25] and [5"]) and the difficulty of precise ~ e l e identification [26] has tempered enthusiasm for their further development and use in genetic mapping and DNA diagnostics. Despite this, VNTRs ~ continue to be used for many applications. For example, the VN'rR locus D I S 8 w a s observed to have > 1070 alleles when the internal core repeats of amplified alleles are scored for the presence or absence of a variable restriction site [23"']. This is an example of a compound polymorphism, which is also seen with STRs (A Edwards and CT Caskey, unpublished results). Satellite DNA found at the primary constriction of chromosomes is known to be highly repetitive. The variability of this satellite DNA has been studied with pulse field gel electrophoresis (PFGE) and long range restriction mapping by using restriction enzymes that rarely cut the satellite DNA, but frequently cut unique DNA. Using these techniques, the human X chromosome cx-sateUite array was shown to vary between 1380 kb and 3730 kb and Mendelian inheritance was observed [10]. These pulse field gel electrophoresis polymorphisms should be useful for linkage to nearby disease genes.
Short tandem repeat polymorphisms Comparison of orthologous DNA sequences and early mutation studies identified tandemly repeated sequences only a few base pairs in length (1-5bp) as regions with higher mutation rates than adjacent single copy sequences [27,28]. It was not until the application of PCR, however, that these markers were successfully studied, and the hypervariability of STRs appreciated [29-33]. The past three years have seen characterization of a large number of dimeric (micro-sateUite), trimeric, and
tetrameric tandem repeats. The relative ease with which they may be amplified, the potential for sequence-based allele identification, uniform dislike across the genome, and their high heterozygosity make them particularly attractive markers. STRs appear to be the third most common class of polymorphic marker found in the human genome, being approximately one half as abundant as A/u repeats. The most frequent STRs a r e dimeric [AC] sequences [34] that are found every 20-30 kb in genomic DNA. Hybridization of five oligonudeotides complementary to specific STRs (e.g., [AGAT]75and [AAT]10) to genomic DNA suggested that trimeric and tetrameric STRs are found once every 300-500 kb [5"] in genomic DNA. As there are 44 unique trirnefic and tetrarneric sequences, the total frequency of trimeric and tetrameric STRs could be as high as one STR per 9 kb; that is, about 200 000 polymorphic STR markers in the human genome. The latter calculation makes the reasonable assumption that 50% or more of the STRs will be polyrnorphic [2",5"]. For unknown reasons, one STR locus may be hyper~-ariable, whereas another locus of the same core sequence and number of repeats may be monomorphic [5"]. It has been shown for dimeric STP,s that loci with greater numbers of repeats (e.g. [AC]20versus [AC]10) are more likely to be polymorphic and have higher heterozygote frequencies [2.]. The data available for trimeric and tetrameric STRs are consistent with the [AC] data [5"]. Incorporating this concept into strategies for identifying randomly distributed polymorphic STRs for genetic mapping and other applications should increase the fracdon of potymorphic STRs to total STRs characterized to more than 75%. There is no evidence that the particular core sequence influences either the probability of a STR being polymorphic or heterozygote frequencies at STR loci are influenced by the particular core sequence. The high heterozygotic and genomic frequencies of STRs has enabled linkage to disease loci in small families [15] and
819
820 Mammaliangene studies the use of an anonymous STR to achieve linkage to the facioscapulohumeral dystrophy locus [16]. Although the hypervafiability and high genomic frequency of [AC]n repeats make them powerful genetic markers, one problem has been interpreting the banding profiles. Generation of repeat length insertion and deletion (presumed slippage) products often creates a 2 bp ladder effect after gel electrophoresis. Additionally, the terminal transferase activity of the thermostable polymerase [35] may convert one or both strands of the 2 b p ladder into a 1 bp ladder if the reaction does not proceed to completion. Consequently, interpretation of the allele profiles may be difficult with particular loci, especially when non-familial samples are being typed. Although the terminal transferase activity is seen with tfimeric and tetrameric STRs, generation of presumed slippage products is much less of a problem [5"], and the longer core motif sequence facilitates allele identification. The amplification fidelity of trimeric and tetrameric STRs has enabled the development of a STR-based DNA typing assay featuring multiplex PCR [36], internal standards and fluorescent detection of alleles [5"]. Most applications of STRs would be facilitated by the availability of methods both to ascertain whether STRs are polyrnorphic apm'om'and to rapidly obtain flanking DNA sequences from which PCR primers could be designed. A reasonable solution to the first problem is selection for trimeric and tetrameric STRs with more than seven repeats. The second problem remains to be solved decisively but a PCR strategy [37 o] for amplifying the ends of YAC inserts using a double stranded oligonucleotide anchor containing an internal region of non-complementarity has been applied to STRs [5"]. By amplifying the sequence between the STR and the non-complementary region of the anchor with primers complementary to the STR and the anchor, DNA segments flanking several [AGAT]n STRs were amplified [5"]. This method has been applied to other STR sequences (I-I~ Hammond, A Edwards, CT Caskey, unpublished results). As experience is accumulated, the utility of the method relative to random strategies [2.] will be determined. Polymorphic trimeric STRs have been found within the coding regions of genes that include the human androgen receptor gene [5"], the Drosophila Notch gene and related genes [32,38], and the gene for the human fragile X locus [39]. Variable tandem reiteration of much longer sequences has also been reported for apolipoprotein A [40] and mucin-type glycoprotein genes [41]. Abnormally high numbers of core repeats (CAG, glutamine) in the hypervafiable trimeric STR within the human androgen receptor genes have been associated with spinal and bulbar muscular dystrophy [14] and other androgen receptor defects [42]. The polymorphic STR within the candidate fragile X locus (CGG, arginine) is involved in the mutations accounting for this disease (Ying Hui Fu et aL, and CT Caskey unpublished results, [39]). These results suggest a strategy' for the identification of disease genes.
Alu polymorphisms The middle repetitive, interspersed Alu repeat occurs approximately 500 000 times in the human genome. It is the most common repetitive sequence in the human genome, occurring on average every 5 kb in genomic DNA. By coupling amplification of Alu repeats in PCRs using primers complementary to unique flanking DNA segments with the recently described technique of singlestrand conformation polymorphism (SSCP) [9",43], approximately 50% of A/u repeats studied were found to be polymorphic [9"]. Heterozygote frequencies ranged from 0.06-0.63 (average 0.38) [9"]. The SSCP method detects polymorphisms by conformational differences observed in single stranded DNA sequences after electrophoresis under non-denaturing conditions, and therefore does not require knowledge of the nudeotide alterations [43]. The use of the SSCP method does, however, require knowledge of the flanking sequence for use with PCIL Most of the A/u polymorphisms detected appear to be single nucleotide substitutions, deletions, or insertions [9o] and thus these polymorphisms may also be incorporated into the automated methods described above. A/u elements are thought to be propagated throughout the genome via retroposition [44]. During this process a poly(A) tail approximately 40 nucleotides long is added to the A/u transcript before insertion into the genome [45]. A cursory review of Alu repeats in GenBank reveals, however, that a significant fraction (6%) of the poly(A) tails become tandemly repeated [(A)nGn] and [(A)nC n] sequences [3]. A fraction of these STRs have been found to be polymorphic, and are useful as genetic markers [3]. in addition to the utility of these sequences as genetic markers, the results imply a mechanism by which other interspersed non-A/u associated STRs may have evolved. A study of the divergence of A/u sequences in genetic databases has led to the subdivision of Alu elements into different classes, thought to be of different genetic ages [46]. The human-specific class is thought to have been transposed in recent human evolution. An oligonucleotide designed to distinguish this class of Alu from approximately 500 000 older repeats enabled the identification of recently transposed sequences in the human genome [8,45,47]. Some of these are insertion polymorphisms for humans, and may provide markers for the study of human evolution.
Arbitrarily primed PCR in the technique of abitrarily primed PCR the amplification is performed with a single arbitrarily chosen primer [48-50]. The amplification products constitute a fingerprint that can be used to distinguish between strains of an organism. The advantage of this technique is that DNA sequence information is not required and it can therefore be applied to any genomic DNA sample for strain identification or other studies.
Genetic marker technology Edwards and Caskey 821 Conclusions
A u-anspositional~" ac~'e A/u subfamily and dimorphismof represenm~ s of this family are described.
Marked advances have been made in our understanding and use of polymorphic genetic markers since the invention of the PCR. Continued application of semi-automated colorimetric and fluorescent detection schemes and multiplexing (the amplification of several markers simultaneously) to STRs should provide the power and Offciency needed for the genetic mapping of large genomes. STRs are also suitable for correlating genetic and physical maps. Because the new mapping approaches are not derivatives of the original two-allele or VNTR polymorphisms, and the development of the new PCR-based markers is proceeding rapidly, it would appear to be more efficient to develop a new set of genetic markers than to continue retrofitting of older, less useful RFLPs. The new polymorphic genetic markers discussed above should provide a rapid method of establishing a genetic map of man and easily used methods for increased the understanding of our genome, its evolution, and gene loC~-liT~qtions.The new PCR-based methods will predictably have wide application in human genetics for an extended period of time.
7.
BAT'ZERMA, DEINI~GER Pt. A Human-Specific Subfamily of
•
Alu Sequences. Genom/cs1991, 9:481-487.
Demonstration of a pob'morphism within the human specific A/u sub-
famib'. 8.
BATT.ER MA, GUDI VA, MENA JC, FOLTZ DW, HERRERA RJ, DEI~2S~GERPL: Amplification Dynamics of Human-Specific (HS) alu Family Members. Nucleic Acids Res 1991, 19:3619-3623.
9. OmTAM, SEKIYAT, HAYASmK: DNA Sequence Polymorphisms • in Alu Repeats. Genom~.s 1990, 8:271-278. A comprehensive study of the types of polymorphisms detected by the sin#e-strand conformation poMnorphismmethod. 10.
MAHTA~aMM, WnIARD HF: Puised.field C~I Analysis of orsatellite D N A at the Human X Chromosome Centromere: High-frequency Polymorphisms and Array Size Estimate. Go nom/cs 1990, 7:607-613.
11.
NICKERSON DA, KAISER P~ LAPPIN S, STEWARTJ, HOOD L,
•
LANDEGRENU: Automated DNA Diagnostics using an ELISA-
based Oligonucleotide Ligation Assay. Proc Nail Acad $ci USA 1990, 87:8923-8927. Description of a potentially automated non-isotopic system for DNA diagnostics using PCR to amplify DNA target segments. 12.
SYVANENA-C, AM.TO-SETALAK, HARJUL, Korqru~ K, SODERLUND H: A Primer-guided Nucleotide incorporation Assay in the Genotyping of Apolipoprotein E. Genom~cs 1990, 8:684-692.
Acknowledgements
13.
A Edwards is a Josephine and Edward Hudson Scholar and Medical S~ntist Training FeUow. CT Caskey is a Howard Hughes Medical Institute Investigator.
CHEHABFF, KAN YW: Detection of Specific DNA Sequences by Fluorescence Amplification: a Color C o m p l e m e n t a t i o n Assay. Prc~ Nail Acad Sci USA 1989, 86.9178--9182.
14.
LA SPADAAR, WILSONEM, lamAia~ DB, HARDINGAE, FISCHBECK KH: Androgen Receptor Gene Mutations in X-linked Spinal a n d Bulbar Muscular Atrophy. Nature 1991, 352:77-79.
15.
HUA~G TH-M, ~ C t K JF, EDWARDS A, P~-nG~EW L H E m ~ CA, HAMMONDHA, CASKEYCT, ZOGHBtHY, I2tmETTER DH: Linkage of the Gene for an X-linked Mental Retardation Disorder to a Hyt~rvariable (AGAT)n Repeat Motif within the Human HPRT Locus (Xq26). Am J Hum Genet 1991, in press.
16.
W[JME~,A C, FRAmS RR, BROU~R OF, MOERER P, WEBER JL, PADBERG GW: Location of Fascioscapulohumeral Muscular Dystrophy Gene o n Chromosome 4. Lancet 1990, 336:651--653.
17.
MYERSRM, LUMELSKYN, LERMANIS, MANIA'mT: Detection of Single Base Substitutions in Total Geneomic DNA. Nature 1985, 313:495--498.
18.
KAN YW, DOZY AM: Pol)maorphism of DNA Sequence Ad. jacent to Human Beta Globin Structural Gene: Relationship to Sickle Mutation. Proc Naa m a d S a USA 1978, 75:5631-5635.
19.
COTIONRGH, RODRIGUESNR, CAMPBELLRE): Reactivity of Cytosine and Thymine in Single-base-pair Mismatches with I-b/droxylamine and Osmium Tcrtroxide and its Application to the Study of Mutations. Proc Nail Acad Sci USA 1988, 85:4397-4401.
20.
SABORK, SCHARFS, FAIDONA F, MULLISIO3, HORN GT, ERLICH HA, /m.'~tzL~ N: Enzymatic Amplification of 13-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cel] Anemia. Science 1985, 230:1350-1354.
21.
NA~MURA Y, LEPPERT M, O'CONNEL P, WOLFF IL HOLM T, CULVERM, M.M~rI~ C, FLrJIMOTOE, HOFF M, KUMLINE, WHn'E l~ Variable Number of Tandem Repeat (VNTR) Markers for Human Gene Mapping. Science 1987, 235:1616-1622.
22.
JEFFREYSAJ, WmSONV, THEIN St- Hypervariable 'Minisatellitc' Regions in Human DNA. Nature 1985, 314.67-73.
References and recommended reading Papers of special interest, published within the annual period of review,
have been hightishtedas: • •.
of interest of outstanding interest
1.
MULUSK, FALOONAF, SCHARF S, SAIKI R, HORN G, EmaCH H: Specific Enzymatic Amplification of DNA in vttr~ the Poiymerase Chain.Reaction. Co/d Spring Harbor Syrup Quant B/o/1986, 51:263-273.
2. ~he
WEBERJt. Informativeness of Human (dCMA)n-(dG-dT)n Polymorphisms. Genom/ca 1990, 7:524-530. author discusses factors that predict whether, or not, a given (AC) repeat will be polymoprhic and the associated heterozygote frequencies. 3.
ECONOMOUEP, BERGEN AW, WARREN AC, ANTONARAKISSE: The Polydeoxyadenylate Tract of Alu Repetitive Elements is Polymorphic in the Human Genome. Proc Natl Acad Set USA 1990, 87:2951-2954.
4.
EPSTEINN, NAHORO, SIXVERJ: The 3' Ends of Alu Repeats are Highly Polymorphic. N u d e ~ Ac/ds Res 1990, 18;4634--4634.
5. •
EDWARDSA, CrerrELLOA, HAMMONDHA, CASKEYCT: DNA TypLag and Genetic Mapping with Trimeric and T e m e r i c Tandem Repeats. Am J Hum Genet 1991, 49:746-757. The genomic frequency and other properties of trimeric and tetrameric tandem repeats in humans is discussed. A method for obtaining DNA sequence flanking STRs and a fluorescent multiplex DNA typing are presented. 6. •
MATERAAG, HE~'~,'N U, SCHMZD CW: A Transpositionally and Trancriptionally Competent Alu Subfamily. Mol Cell Biol 1990, 10:5424-5432.
822
Mammalian g e n e studies JEFFRE~AJ, NEUMANNR, WI~ON V: Repeat Unit Sequence Variation in MinisateUites: a Novel Source of DNA Polymorphism for Studying Variation and Mutation by Single Molecule Analysis. Cell 1990, 60:473-485. Descriptjo' n of an elegant assay for DNA typing and study of the evolution of VN'IP,s. Jeffre~ et al provide direct estimates for the mutation rate of a VNTR locus and describe the structure of the mutated aUeles.
Description of an anchored-PCR method ~ith significant ack~ntages over other methods. 38.
WHARTONK, YEDVOBN1CKB, FtNNEtrrYV, ARTAVANIS-TSAKO.',~ASS: opa: A Novel Family of Tr-an~ril~d Repeats Shared by the Notch Locus and Other Developmentally Regulated Loci in D. melanogaster. Cell 1985, 40:55-62.
24.
JEFFREY5AJ, WILSONV, NEUMANNK KEYIXJ: Amplification of Human Mini-satellites by the Polymerasc Chain Reaction: Towards DNA Fingerprinting of Single Cells. Nucleic Acids Res 1988, 16:10953-10971.
39.
25.
ARMOURJA, POVEYS, JEREMIAHS, JEFFREYSAJ: Systemic Cloning of Human Mini-Satellites from Ordered Array Charomid Libraries. Genom/cs 1990, 8:501-512.
VERk~RKAJMH, PmRETn M, SUTO.WFEJS, FU Y-H, KUHL DPA, P1ZZUTI A, RELNERO, RICHARDSS, VICTORIAME, ZHANG F, ET at.: Identification of a Gene (FMR-1) containing a CGG Repeat Coincident with a Breakpoint Cluster Region Exhibiting Length Variation in Fragile X Syndrome. Cell 1991, 65:905-914.
40.
KOSCH~SKYM~ BELSIEGELU, HENNE-BRUNSD, EATONDL, LAWN RM: Apolipoprotein A Size Heterogeneity is Related to Variable Number of Repeat Sequences in its mRNA. B ~ e r n ~try 1990, 29:640-644.
41.
SWALLOWDM, GENDLER S, GRtrrnvLS B, CO~XY G, TAYLORPAPADIMrrRIouJ, BRAMWELLME: The Human Tumour-associated Epithelial Mucins are Coded by an Expressed Hypcr,,-ariable Gene Locus PUM. Nature 1987, 328:82-84.
42.
MCPHAULMJ, MARCELU M, Tnazv WD, GRtFF~ JE, ISlImOGmaERREZ RF, WILSONJD: Molecular Basis of Androgen Resistance in a Family with a Qualitative Abnormality of the Androgen Receptor and Responsive to High-dose Androgen Therapy. J Clin Invest 1991, 87:1413-1421.
43.
OmTA M, SUZUKl Y, SEKIYA T, tIAYASm K: Rapid and Sensitive Detection of Point Mutations and DNA Polymorphisms using the Polymerasc Chain Reaction. Genomica 1990, 5:874-879.
44.
ROGERSJ: Retroposons Defined. Nature 1983, 301:460-460.
45.
BATZERMA, KILROYGE, ~CHARD PE, SHAtKHTH, DESSELLETD, HOPPENS Cl~ DEL~q2qGERPL: Stl~cture and Variability of Recently Inserted Alu Family Members. Nucleic Acids Res 1991, 18:6793-6798.
46.
SLAGELV, FLEMLNGTON E, TRA~A-DORGE V) BRAI~HAW H, DE1NLNGERPI2 Clustering and Subfamily Relationships of the Alu Family in the Human Genome. Mo/ B/o/ EL~/ 1987, 4:19-29.
47.
MArERAAG, HEILMANN U, HINTZ MF, SCHMID CW: Recently Tt-ansposed Alu Repeats Result from Multiple Source Genes. Nucleic Acids Res 1991, 18:6019--6023.
48.
WIILIAMSJGK, KUBELIK AR, LWAK KJ, RAFALSKIJA, "I~GEY SV; DNA Polymorphisms Amplified by Arbiwary Primers are Useful as Genetic Markers.. Nucleic Acids Res 1990, 18:6531-6535.
49.
WELSHJ, McCLELUe~ M: Fingerprinting Genomes Using PCR with Arbitrary Primers. Nucleic Acids Res 1991, 18:7213-7218.
50.
WELSHJ, P~XRSEN C, MCCLEtL~','DM: Polymorphisms Generated by Arbitrarily Primed PCR in the Mouse: Application to Strain Identification and Genetic Mapping. Nucleic Acids Res 1991, 19:303-306.
23. **
26.
DEvI~ B, R1SCH N, ROEDER K: No Excess of Homozygosity at Loci Used for DNA Fingerprinting. Sc/ence 1990, 249:1416-1420.
27
SAVATIERP, "I"~d3UCHETG, FAURE C, CHEBLOUNEY) GODY M, VERDIER G, NIGON VM: Evolution of the Primate ~-Globin Gene Region: High Rate of Variation in CpG Di.nucleotides and in Short Repeated Sequences between Man and Chimpanzee. J Mo/B/cd 1985, 182:21-29.
28,
SEMENZAGL, MAUADI P, SURREY S, DELGROSSO K, PONCZ M, SCHWAR'IZ E: Detection of a Novel DNA Polymorphism in the 13-globin Gene Cluster. J Biol O~em 1984, 259:6045-6048.
29.
WEBERJL, MAY PE: Abundant Class of Human DNA Polymorphisms which can be Typed Using the Polymerasc Chain Reaction. Am J Hum Genet 1989, 44:388-396.
30.
Lrrr M, U.rry JA: A Hypervarlablc Microsatellite revealed by in vitro Amplification of a Di-nucleotidc Repeat Within the Cardiac Muscle Actirt Gene. Am J Hum Genet 1989, 44 -.397---401.
31.
Dm0A "I'P, MUKAI S, PETERSON R, RAPAPORTJM, WALTON D, YANDELLDW: Parental Origin of Mutations of the Retinoblastoma Gene. Nature 1989, 339:556-558.
32.
TALrm D: Hypcrvarlabilityof Simple Sequences as a General Source for Polymorphic D N A Markers. Nu~eic Acids Res 1989, 17:6463-6471.
33.
EDWARDS A, GraBS RA, NGUYEN PN, ANSORGE W, CASKEY CT: Automated D N A Sequencing Methods for Dctcction and Analysis of Mutations: Applications to the Lesch-Nyhan Syndrome. Tram Assoc Am Physiciam 1989, CII:185--194.
~P~.
STAIMNGSR]., FORD AF, NELSON D, TORNEY DC, HOLDEBRA.ND CE, MoYzls RK: Evolution and Distribution of (GT)n RepetRive Sequen~:es in Mammalian Genomes. Genom/ca 1991, 10:807-815.
35.
CLARKJM: Novel Non.tcmplated Nucleodde Addition Reactions Catalyzed by Prokaryotic and Eukaryotic DNA Polymer. Nuc/e~ Adds Res 1988, 16:9677-9686.
36.
CHAMBERtA~JS, GraBS RA, RANTERJE, NGUYENP-N, CASKEYCT: Deletion Screening of the Duchcnne Muscular Dystrophy Locus via Multiplex DNA Amplification. Nuc/e/c Adds Re$ 1988, 16:11141-11156.
37.
Rn.EYJ, BUTLERR, OGILVIE D, FINNmAR R, JENNER D, POWELL
*
S, ANAND R, SMITHJC, MARKHAMAF: A Novel Rapid Method
for the Isolation of Terminal Sequences from Yeast Artitidal Chromosome (YAC) Clones. Nucleic Acids Res 1990, 18:2887-2890.
A Edwards and CT Caskey, Institute for Molecular Genetics, and Howard Hughes Medical Institute, Baytor College of Medidne T809, 1 Baylor Plaza Houston, Texas 77030, USA.
