THE INTERNATIONAL FEDERATION CLINICAL CHEMISTRY
OF
IFCC Section (1992) Recent IFCC Publications
Published by ELSEVIER SCIENCE PUBLISHERS on behalf of THE INTERNATIONAL FEDERATION OF CLINICAL
CHEMISTRY
s2
The exclusive 0 for all ian~a~s F~e~tion of Clinical Chemistry.
and countries is vested in The I~te~a~ona~
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior permission of the copyright okner.
List of IFCC I’ubliixtions IFCC Sections (1978) No. I No. 2
Clin. Chim. Acta (19’78) Vol. 83, 189F-202F Vol. 87,459F-465F
IFCC Sfxtions (1979) No. 1 No. 2 No. 3
Clin. Chim. Acta (1979) Vol. 95, 153F-188F Vol. 96, 155F-204F Vol. 98, 127%186F
IFCC Se$ians (1980) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1980) Vol. lO3,239F-258F Vol. 105, 147F-172F Vol. 106, IWF-120F Vol. 108,4#F-539F
IFCC Section (1981)
Clin. Chim. Acta (1981) Vol. 109, 103F-124F
IFCC Sections (1982) No. 1 No. 2
Clin. Chim. Acta (1982) Vol. 119, 351F-362F Vol. 122, 109F-123F
IFCC Sectiizms (1983) No. 1 No. 2 No. 3 No. 4 No. 5
Clin. Chim. Acta (1983) Vol. 127,423F-448F Vol. 129, 2453-249F Vol. l31,349F-359F Vol. 134, 369F-386F Vol. 135, 313F-367F
IFCC S&ians (1984) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1984) Vol. 137, 95F-114F Vol. 137, 369F-386F Vol. 139, 203F-230F Vol. 143, 69F-72F
IFC No. No. No. No.
Clin. Chim. Acta (1987) Vol. 165,95-l 18 Vol. 170, 53 Vol. 170, 513 Vol. 170, s33
Sections (1987) 1 2 3 4
s3 IFCC Sections (1988) No. 1 No. 2
Clin. Chim. Acta (1989) Vol. 185, Sl Vol. 185. S17
IFCC Sections (1991) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1991) Vol. 202, Sl-s12 Vol. 202, S13-S22 Vol. 202, S23-MO Vol. 203, Sl-S16
IFCC Sections (1992) No. 1 No. 2 No. 3 No. 4 No. 5 No. 6
Clin. Chim. Acta (1992) Vol. 205, Sl-S15 Vol. 205, Sl7-S23 Vol. 207, Sl-Sll Vol. 207, S13-S26 Vol. 209, Sl-s13 Vol. 209, Sl5-S26
SS
CIinica Chimica Ada, 209 (1992) SS-S 13 Elsevier Science Publishers B.V. 0009-8981/92/$05.00 CCA 05346
INTERNATIONAL
FEDERATION
OF CLINICAL CHEMISTRY
Diagnostic applications of renetitive DNA sequences J&-g T. Epplen Scienti@ Division Committee on Molecular Sioiogy Techniques
Key words: Genomic redundancy; DNA profiling; Multilocus systems: Man and animals: Plants; Fungi
The potential, the advantages and the different areas of diagnostic applications are discussed for the various categories of repetitive DNA sequences. Since all eukaryotes are characterized by genomic redundancy, these sensitive, rapid and comparatively simple techniques are revolutionizing many a field of clinical and experimental diagnostics.
Most eukaryotic genomes are several orders of magnitude larger than those of prokaryotes. The reason for this vast abundance of genetic material has initially been interpreted in the context of the seemingly larger complexity and abilities of the eukaryotes. The true reasons, however, may be more closely related to a certain mode of evolutionary strategy pursued by the eukaryotic species: Eukaryotes rather tend to preserve extensively large genomes to allow comparatively quick changes in the’interactions of individual (parts of) genes and their regulatory units. Hence novel interrelations of the genetic elements and changes in the regulation of gene expression could apparently permit increased versatility if the organisms are exposed to novel environmental conditions. In contrast prokaryotes tend to ‘streamline’ their genomes to the absolute minimum size in order to attain utmost rapid multiplication. Their potential to evolve successfully is therefore almost exclusively limited to Correspondence to: Jiirg T. Epplen, Molekulare Humangenetik der Ruhr-Universitit IO 21 48, W-4630 Bochum 1, FRG. The copyright is vested in IFCC. April 92. S.D.-CMBT13
Bochum. Postfach
(adaptive) point mutations. Yet certainly not all of the potential genetic information of eukaryotes can harbor a sequence-dependent functional meaning. Otherwise even the naturally caused mutation load were impossible to bear for them. Thus survival of the eukaryotic organisms were highly unlikely in the past and present environmental conditions. In eukaryote genomes a number of different types of DNA sequences have been identified in the genomic ‘desert’ between the ‘oases’, i.e. the genes. In addition to the prevailing single copy elements (one copy per haploid genome), various kinds of redundant, repetitive sequences constitute a major part of this so-called ‘secondary DNA’ [l] In general the surplus redundant material is not transcribed si~i~cantly and the transcripts are translated only exceptionally (see Ref. 2). Exceptions that confirm the rule are ribosomal RNA encoding genes, histon genes and a few more large gene families. Nevertheless most repetitive sequences do certainly neither contribute to the phenotype nor to the behaviour of the organisms. In consequence most of the repetitive DNA sequences have been regarded as ‘junk and jumble’ of the evolutionary past in the respective species 131. Yet irrespective if these sequences should be categorized as ‘innocent’, ‘ignorant’, ‘illusive’, ‘selfish’ or ‘parasitic’ (for a discussion of this semantic subject see Refs. 4 and S), their major present-day meaning can be seen in their successful applications as handy tools for various diagnostic endeavours [6]. There are several types of repetitive elements, among which five pr~ominant classes emerged quite consistently in the eukaryote genomes studied intensively so far (for review see e.g. Ref. 7). Variable number of tandem repeats (VNTR) have in common that their basic unit is repeated several to many times in a head,-to-tail fashion, VNTR should be subclassified into: (A) Simple repeat sequences and [8] and (B) the more complex ~j~~sa~e~~i~es 19,101. Simple repeats consist of short sequence motifs up to 10 bases repeated over and over in a tandem orientation (the upper length limit of the motifs in this category is still discussed). The minisatellites have a longer periodicity due to the larger basic core sequence. They could well have developed from initially perfect simple repeats by sequence divergence during evolution, additional amplification and secondary redistribution. (C) Short interspersed ~uc~earideelements (‘SINES’) are less than 500 base pairs long. Eminent examples exist like the well-known Alu sequences in man and the evolutionarily related Bl elements in the mouse. These repeat elements are quantitatively prevalent in many animal (and plant) genomes with up to millions of copies distributed all over the chromosomal complement though not completely randbm (for comprehensive review and definitions see Ref. 11). (D) i,ong interspersed ~uc~ea~ideelements (‘LINES’) harbor several kilobases of length and show often characteristic signs of retroposons [12]. (E) Extremely long runs of simple repeats (see A) or their degenerated, sequence divergent spin-offs constitute classical satellite DNA, which can in most cases be separated physically from the bulk of the remaining genome by bouyant density gradient cent~fugation. Using cytogenetic staining methodolo~, this material can often be visualized as so-called constitutive heterochromatin (see Ref. 13). Large chunks of this particular type of DNA are sometimes accumulated on the sex
s7
chromosomes of many (animal) species in which location it apparently does neither harm nor matter at all. Among these DNA sequences in man the cu (alphoid) and /3 satellites have been characterized in extenso [ 141. Also in this alphoid DNA a core motif of about 171 base pairs in length could be identified. Until now the full functional or biological meaning of most untranscribed and untranslated repetitive DNA sequences remained completely obscure. Nevertheless repetitive DNA has been employed as versatile tools in many areas where molecular biology techniques are being utilized. The following statements represent a listing of various diagnostic fields, where DNA probes containing repetitive sequence elements are currently already being applied successfully. Individuality Testing including Analyses in the Forensic Sciences Tools
A4ultilocus probes such as hypervariable minisatellites [9, lo], Ml 3 phage derived sequences (151, simple repetitive sequences [6,16,17] are freely available (though patented for commercial purposes) and sufficiently well characterized in the human and many animal and plant genomes. Probes are either molecularly cloned or synthetic oligonucleotides. in addition cloned and/or synthetic oligonucleotide probes specific for individual hypervariable loci have been developed which contain (either repetitive or more often) single copy sequences (allele specific oligonucleotides ‘ASOs’; for a compilation see Ref. 18). These markers are usually investigated and demonstrated by blot or direct in-gel hybridization. The latter procedure avoids the necessity for time-consuming blotting. In the last several months a wealth of highly informative simple repeal polymorphisms (SRPs; sometimes called ‘microsatellites’) has been published (as of today close to 300). Many more are available in individual laboratories. These DNA loci contain mostly (ca),/(gt), or related short simple sequences. They are localized in the genomic spacer regions (between genes), in 5 ‘-and 3 ‘-untranslated parts of messages as well as in introns of genes. Already all human chromosomes are covered in close to sufficient ‘density’ to map the complete human genome physically by SRPs. (Though human Y chromosomal loci have not yet appeared in press, we have characterized several of them in the Y-specific and the pseudoautosomal regions [19]). The major advantage of SRPs concerns the extreme degree of informativity (polymorphism information content, PIC) and an exactly definable chromosomal localization of these mulrial~elic markers as well as the speed with which they can be generated from chromosome-specific or ordered cosmid libraries and applied to all kinds of studies based on the linkage concept. For many of the SRPs, data on the somatic and meiotic mutation rates, etc. have yet to be established. Techniques
Multilocus fingerprints can be produced on Southern-blotted DNA samples (see above [9,10]) or preferably directly in situ in the gel [2U]. In general single copy DNA loci are probed from conventional agarose gels (mostly blotted onto supporting
membranes) or after amplification by polymerase chain reaction (PCR) of individual hypervariable loci on agarose or preferably on high resolution polyacrylamide or even denaturing sequencing gels. The abovementioned locus-specific SRPs are amplified by PCR and they can also be analyzed gelelectrophoretically. Applications
(a) Typing/matching for transplantation purposes (bone marrow, kidney, heart, liver, lung, etc.), zygosity determination in twins, detection of somatic mosaicism and chimerism, monitoring of graft-versus host reactions after blood transfusions (see e.g. Refs. 21 and 22); (b) animal and plant species and/or individual identification [6,9- 10,17,23], including mixing of blood samples, burnt bodies, parts of bodies [24); (c) paternity and/or family relationships, also possible in incest and deficiency cases [9,10]; (d) identification of stains [25J; (e) identification of cultured cells [26,27]; (f) studies in the behavioural sciences [27a,b]. Advantages
in comparison
to conventional
and other methods
For routine individualization cases (e.g, paternity), DNA fingerprinting using multilocus probes represents a quick, cheap and technically comparatively simple technique which is several orders of magnitude more informative with respect to individualization than other approaches (bloodgroups and other serology, HLA typing, obsolete phenotypic trait comparison procedures). One single analysis is sufftcient as compared to the multiple different experimental steps involved for determining the polymorphisms of serum enzymes etc. In the meantime the formerly disputed statistical background for the probability calculation of paternity with multilocus probes has largely been settled [28-301 and also cases with mutations in the offspring and incest as well as deficiency cases (incomplete trios) can be treated with the appropriate statistics. If necessary the sensitivity in stain analysis can be scaled up to the theoretical maximum by PCR, i.e. to demonstrate one single molecule (cave the ubiquitous danger of contaminations, which is intrinsic to the technique of the PCR but never discussed and appreciated appropriately). The simultaneous PCR of 3-5 highly informative SRP loci renders sufficiently differentiating individualization probabilities in most instances [ 191. Determhation
of Sex
in MM,
Animals ad Plants
Tools
Preferably Y chromosomal repeats are used, e.g. the Ha&I-produced 2.1 or the 3.4 kilobase repeat sequences. It is also possible to employ single copy sequences though naturally with reduced sensitivity [31,32]. Also birds (reptiles, fish and plants?) can be sexed by repetitive W- or Y-chromosomal sequences present only in the females (ZUZW sex chromosome mechanism) or the males (XXXY system).
s9
Techniques Direct hybridization to Southern-blotted, genomic DNA or PCR amplified material is most often appropriate 1331. It is even possible to start from maternal blood since fetal cells cross the placenta (see e.g. for single copy sequences [34]) Previous pregnancies with male fetuses have to be accounted for since lymph~ytes may last for years undetected by the immune system in the maternal circulation. {a) Demonstration of Y chromosomal DNA from blood, all other tissues and stains; (b) prenatal diagnosis for X-chromosomally inherited diseases; (c) preimplantation screening with single cells of the embryo; (d) sperm diagnosis; (e) animal and plant breeding (see below). Advantages in comparison fo other melhods This is a fast and simpk procedure since cell culture is not necessary. Theoretically the PCR is even possible from single cells obtained, e.g. from preimplantation em-. bryos or from blood of the pregnant mother (cave: ~ontaminat~ons~ see above and below). Diagnosis of Infectious Agents (Eukaryotic Organisms like Ciliates, Fungi, Protozoa, e.g. LRiskmaniaad Trypanosoma Species)
Simple repetitive ohgonucleotide
sequences.
Techniques Preferably by in-gel hybridization, Southern blot hybridizations are less advisable. {a) Species/strain identification; (b) differentiation of pathogenetic versus nonpathenogenetic strains of one species, e.g. in fungi and ciliates; (c) monitoring of genomic changes in the genomes of the infectious agents during the course of the
disease/infection. Advantagescompared to other methods The fast and simple (sub-) classification procedure is possible without extensive culture of the agents and without any laborious biochemical analysis of the organisms (if at all possible using non-fingerprinting methods). For several of the parasites biochemical marker analyses ,have not even been described. Yet even pathotyping in fungi has already been established [35]. Tumors Tools
The whole collection of different simple repetitive ohgonucleotide
sequences can
be used efficiently for a genera1 ‘genome scanning’ approach. Since the minisatellites and Ml3 target sequences are preferentiaily Iocalized only on the telomeres, they are sometimes less informative. Techniques See Individuality testing.. .. (a) Determination of clouality and of the relationship of primary tumors to metastases; verification of secondary independent tumor development; (b) screening for DNA amplification events; (c) changes in the DNAmethylation status; (d) monitoring of chromosomal losses and rearrangements; (e) and other chromosomal aberrations (duplications, partial deletions, mono/trisomies, etc.). Advantages compared to other methods Using approximately ten different oligonucleotide probes this sensitive, simultaneous and simple detection method achieves the analysis of many scattered DNA loci across the whole genome (the whole chromosomal complement). In Situ Hybridi&on/Chromo
Pninting in Man and Anh~ls
Tools
See Individuality resting . ... In addition different classical satellite sequences are useful for particular purposes. Techniques See Individuality testing .. . . The target DNA is detected directly on microscopic slides, preferably with non-radioactive reporter groups such as fluorochromes, biotin, digoxigenin or directly to the probe coupled alkaline phosphatase. The last version of probes is less advisable because of possible steric hindrances during hybridization. (a) Localization of individual repeat sequences on metaphase chromosomes; (b) identification of centromeres by alphoid satelIite sequences; (c) identification of nucleolus organizer regions (NORs) by 6 satellites or interspersed simple repeats; (d) repeat sequence ‘architecture’ in interphase nuclei; (e) histologic preparations. Advantages in comparison to other methods These include the higher sensitivity as compared to single locus probes due to the repetitive nature in a given locus and/or many hybridization events at different sites. Therefore also non-radioactive approaches are possible which are faster (signal developing time) and more precise in the subcellular microscopic localization (no range of the signal like with e-irradiation, e.g. from 32P-, “S-label or from [3N]thymidine). Most importantly no special precautions for radioactivity are necessary.
Animal (nnd plant) Ikediug
See individuality testing . ... Different fields of applications are open for the different multilocus probes (minisatellites; Ml3 sequences and simple repeats). As of today the largest spectrum of species (> 200 fungi, plants and animals} seems accessible by using simple repetitive oligonucleotide probes. Techniques See Individualify testing . .. . (a) De termination of paternity, maternity, zygosity and other genetic relationships for behavioural or socio-biological and experimental animal studies; (b) meat and animal identification (also for forensic purposes); (c) delineation of quantitative traits in breeding experiments; (d} efficient .inbreeding control for farm and/or laboratory animals; (e) determination of genetic relationships in endangered species (e.g. zoo or wild life animals) for selective breeding in order to maintain the gene pools as large as possible and therefore maximum heterozygosity; (f) identi~cation of the sex in species, where it is not possible without life endangering methods, like endoscopy for birds; (g) identification of cultured cells and contaminations therein. Advantages
compared to other techniques
DNA fingerprinting is highly informative for individualization. It is performed rapidly and technically simple and in addition also comparatively cheap. One principal technique is sufftcient for all aforementioned applications. Depending on the probe more or less the whole genome can be covered with interspersed simple repeat loci. Enformative simple repetitive oligonucleotide probes are available for all species tested so far. Genome Mapping and Aaalytis Tools
In order to characterize and to map large (parts of) eukaryotic genomes recently several simple repetitive nucleotide repeat sequences have been employed (mainly dinucleotide SRPs like (ca}J(gt)n or (ct)J(ga),; see above under Individuality testing ._.). The longer the perfect simple repeat stretches are, the more variable and hence informative are these DNA loci. Upon flanking DNA sequence analysis two antiparallely orientated primers are synthesized on both sides of the repeat and the standard PCR reaction is performed. Techniques
Amplification by PCR and gel electrophoretic analysis by staining or probe hybridization according to conventional protocols. Cave contaminations, especially
when the amount of the sample analyzed is very small (only few molecules from stains); therefore many appropriately designed contamination controls have to be performed routinely. Applications The procedure allows the ordering of genomic phage, cosmid and yeast artificial chromosome (YAC) libraries into long physically coherent DNA (chromosome) segments, the so-called contigs. Large scale genomic mapping projects. Linkage analysis to (disease) genes. Advantages in comparison to other and conventional methods The high informativity in comparison to conventional restriction fragment length polymorphisms (RFLPs) renders it a rapid and eficient analysis of many samples in very short time. In addition the number of necessary investigations (informative families) can be reduced considerably in order to firmly establish e.g. genetic linkage of a marker locus to a disease gene. References 1 Hinegardner R. Evolution of genomt size. In: AyaJa FJ. ed. Molecular evolution. Sunderland: Sinauer, 1976;179- 199. 2 L&bold DM, Swergold GD, Singer MF, Thayer RE, Dombroski BA. Fanning TG. Translation of LINE-DNA elements in vitro and in human cells. Proc Nat1 Acad Sci USA l!I90;87:6990-6994. 3 Uhno S. So much junk in our genome. Brookhaven Symp Biol 1972;23:366-370. 4 ‘Mobile DNA’. in: Berg and Howe, ed., Wiley and Sons, 1989. 5 Epplen JT. On simple repeated GATAIGACA sequences in animal genomes: A critical reappraisal. 3 Hered 1988;79:409-417. 6 Epplen JT, Ammer H, Epplen C et al. Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes. In: Burke G, Dolf G, Jeffreys AJ. Wolff R. eds. DNA fingerprinting: Approaches and applications. Basel: Birkhiiuser, ~99150-69. 7 Studer R, Epplen JT. On the organization of animal genomes; ubiquitous interspersion ofrepetitive DNA sequences. in: Geldermann H. Ellendorff F, eds. Genome analysis in domestic animals. Wcinheim/New York: VCH-Veriag, 1990;17-33. 8 Tautz D, Rent M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Rcs 1984;12:4f 27-4 I 38. 9 Jeffreys AJ, Wilson V, Thein SL. Hypervariable “minisatellite” regions in human DNA. Nature 1985a;314:67-73. 10 Jeffrcys AJ, Wilson V, Thein SL. Individual-specific fingerprints of human DNA. Nature 1985b;316:76-79. 11 Singer MF. SINES and LINES: Highly repeated short and long interspersed sequences in mammalian genomes. Cell 1982;28:433-434. 12 Rogers J. Retroposons defined. Nature 1983;301:460. 13 Advanced techniques in chromosome research. Adolph KW, ed. New York: Marcel Dekker 1991;1-462. 14 Waye JS, Willard HF. Human #3satellite DNA: genomic organization and sequence definition of a class of highly repetitive tandem DNA. Proc Natl Acad Sci USA 1989$6:6250-6254. IS Vassart G, Georges M. Monsieur R. Brocas H, Lequarre AS, Christophe D. .4 sequence in M I3 phage detects hypervariable minisatellites in human and animal DNA. Science 1987;235:683-684. 16 Ali S. Miiller CR, Eppien JT. DNA fingerprinting by oligonucleotides specific for simple repeats. Hum Genet 1986;74:239-243.
s13 17 Weising K, Ramser J, Kaemmer D, Kahl G, Epplen JT. Oligonucleotide fingerprinting in plants and fungi. In: Burke T, Dolf G, Jeffreys AJ, Wolff R, eds. DNA fingerprinting: Approaches and applications. Basel: Birkhiiuser, 1991;312-329. 18 Human Gene Mapping Library, Yale University/Howard Hughes Medical Institute, New Haven, Connecticut. 19 Roewer L, Arnemann J, Spurr NK, Grzeschik K-H, Epplen JT. Simple repat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts. Hum Genet in press. 20 Tsao SGS, Brunk CF, Pearlman RE. Hybridization of nucleic acids directly in agarose gels. Anal Biochem 1983;131:365-372. 21 Ugozzoli et al. Genotypic analysis of engraftment in thalessemia following bone marrow transplantation using synthetic oligonucleotides. Bone Marrow Transpl 1989;4:I73- 180. 22 Kunstmann E, Backer T, Sauer H, Mempel W, Epplen JT. Diagnosis of transfusion-associated graft-versus-host disease by genetic fingerprinting and polymerase chain reaction. Transfusion, in press. 23 Niirnberg P, Roewer L, Neitzel H et al. DNA fingerprinting with the oligonucleotide probe (CAC)s/(GTG),: somatic stability and germline mutations. Hum Genet 1989;84:75-78. 24 Piiche H, Wrobel G, Schneider V, Epplen JT. The identification of a charred body by oligonucleotide fingerprinting with the (GTG)5 probe. In: Berghaus G, Brinkmann B, Rittner C, Staak M, eds. DNA-technology and its forensic application. Heidelberg: Springer-Verlag, 1991;192-195. 2.5 Roewer L, Niirnberg,P, Fuhrmann E, Rose M, Prokop 0, Epplen JT. Stain analysis using oligonucleotide probes specific for simple repetitive DNA sequences. Forensic Sci Int 1990,47:59-70. 26 Gilbert DA, Reid YA, Gail MH et al. Appli~tion of DNA ~ngerp~nts for cell-line individualization. Am J Hum Genet 1990,47:499-514. 27 Speth C, Epplen JT, Oberbaumer I. DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor. In Vitro Cell Dev Biol 1991;27A:646-650. 27a Burke T, Davies NB, Bruford MW, Hatchwell BJ. Parental care and mating behaviour of polyandrous dunnocks Pruneita modularis related to paternity by DNA fingerprinting. Nature 1989;338:249-251. 27b Achmann R, Heller K-G, Epplen JT. Last male sperm precedence in the bushcricket Poecittimon vetuchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting. Mol Ecol 1922;in press. 28 Evett IW, Werrett DJ, Buckleton JS. Paternity calculations from DNA multilocus profiles. J Forensic Sci Sot 1989;29:249-254. 29 Yassoilridis A, Epplen JT. On paternity determination from multilocus DNA profiles. Electrophoresis 1993;12:221-226. 30 Krawczak M, Biihm I, Nib&erg P, et al. Paternity testing with probe (GTG)~/(CAC)~: a multicenter study. Hum Genet 1992; in press. 31 Page DC, Mosher R, Simpson EM et al. The sex-determining region of the human Y chromosome encodes a finger protein. Cell I987;51:1091-1104. 32 Sinclair AH, Berta P, Palmer MS et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990;346:240-244, 33 Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768-770. 34 Ebensperger C, Studer A, Epplen JT. Specific amplification of the ZFY gene to screen sex in man. Hum Genet 1989;82:289-290. 35 Weising K, Kaemmer D, Epplen JT, Weigand F, Saxena M, Kahl G. DNA fingerprinting of Ascochylu rabiei with synthetic oligodeoxynucleotides. Curr Genet 1991;19:483-489.
OF
IFCC Section (1992) Recent IFCC Publications
Published by ELSEVIER SCIENCE PUBLISHERS on behalf of THE INTERNATIONAL FEDERATION OF CLINICAL
CHEMISTRY
s2
The exclusive 0 for all ian~a~s F~e~tion of Clinical Chemistry.
and countries is vested in The I~te~a~ona~
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior permission of the copyright okner.
List of IFCC I’ubliixtions IFCC Sections (1978) No. I No. 2
Clin. Chim. Acta (19’78) Vol. 83, 189F-202F Vol. 87,459F-465F
IFCC Sfxtions (1979) No. 1 No. 2 No. 3
Clin. Chim. Acta (1979) Vol. 95, 153F-188F Vol. 96, 155F-204F Vol. 98, 127%186F
IFCC Se$ians (1980) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1980) Vol. lO3,239F-258F Vol. 105, 147F-172F Vol. 106, IWF-120F Vol. 108,4#F-539F
IFCC Section (1981)
Clin. Chim. Acta (1981) Vol. 109, 103F-124F
IFCC Sections (1982) No. 1 No. 2
Clin. Chim. Acta (1982) Vol. 119, 351F-362F Vol. 122, 109F-123F
IFCC Sectiizms (1983) No. 1 No. 2 No. 3 No. 4 No. 5
Clin. Chim. Acta (1983) Vol. 127,423F-448F Vol. 129, 2453-249F Vol. l31,349F-359F Vol. 134, 369F-386F Vol. 135, 313F-367F
IFCC S&ians (1984) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1984) Vol. 137, 95F-114F Vol. 137, 369F-386F Vol. 139, 203F-230F Vol. 143, 69F-72F
IFC No. No. No. No.
Clin. Chim. Acta (1987) Vol. 165,95-l 18 Vol. 170, 53 Vol. 170, 513 Vol. 170, s33
Sections (1987) 1 2 3 4
s3 IFCC Sections (1988) No. 1 No. 2
Clin. Chim. Acta (1989) Vol. 185, Sl Vol. 185. S17
IFCC Sections (1991) No. 1 No. 2 No. 3 No. 4
Clin. Chim. Acta (1991) Vol. 202, Sl-s12 Vol. 202, S13-S22 Vol. 202, S23-MO Vol. 203, Sl-S16
IFCC Sections (1992) No. 1 No. 2 No. 3 No. 4 No. 5 No. 6
Clin. Chim. Acta (1992) Vol. 205, Sl-S15 Vol. 205, Sl7-S23 Vol. 207, Sl-Sll Vol. 207, S13-S26 Vol. 209, Sl-s13 Vol. 209, Sl5-S26
SS
CIinica Chimica Ada, 209 (1992) SS-S 13 Elsevier Science Publishers B.V. 0009-8981/92/$05.00 CCA 05346
INTERNATIONAL
FEDERATION
OF CLINICAL CHEMISTRY
Diagnostic applications of renetitive DNA sequences J&-g T. Epplen Scienti@ Division Committee on Molecular Sioiogy Techniques
Key words: Genomic redundancy; DNA profiling; Multilocus systems: Man and animals: Plants; Fungi
The potential, the advantages and the different areas of diagnostic applications are discussed for the various categories of repetitive DNA sequences. Since all eukaryotes are characterized by genomic redundancy, these sensitive, rapid and comparatively simple techniques are revolutionizing many a field of clinical and experimental diagnostics.
Most eukaryotic genomes are several orders of magnitude larger than those of prokaryotes. The reason for this vast abundance of genetic material has initially been interpreted in the context of the seemingly larger complexity and abilities of the eukaryotes. The true reasons, however, may be more closely related to a certain mode of evolutionary strategy pursued by the eukaryotic species: Eukaryotes rather tend to preserve extensively large genomes to allow comparatively quick changes in the’interactions of individual (parts of) genes and their regulatory units. Hence novel interrelations of the genetic elements and changes in the regulation of gene expression could apparently permit increased versatility if the organisms are exposed to novel environmental conditions. In contrast prokaryotes tend to ‘streamline’ their genomes to the absolute minimum size in order to attain utmost rapid multiplication. Their potential to evolve successfully is therefore almost exclusively limited to Correspondence to: Jiirg T. Epplen, Molekulare Humangenetik der Ruhr-Universitit IO 21 48, W-4630 Bochum 1, FRG. The copyright is vested in IFCC. April 92. S.D.-CMBT13
Bochum. Postfach
(adaptive) point mutations. Yet certainly not all of the potential genetic information of eukaryotes can harbor a sequence-dependent functional meaning. Otherwise even the naturally caused mutation load were impossible to bear for them. Thus survival of the eukaryotic organisms were highly unlikely in the past and present environmental conditions. In eukaryote genomes a number of different types of DNA sequences have been identified in the genomic ‘desert’ between the ‘oases’, i.e. the genes. In addition to the prevailing single copy elements (one copy per haploid genome), various kinds of redundant, repetitive sequences constitute a major part of this so-called ‘secondary DNA’ [l] In general the surplus redundant material is not transcribed si~i~cantly and the transcripts are translated only exceptionally (see Ref. 2). Exceptions that confirm the rule are ribosomal RNA encoding genes, histon genes and a few more large gene families. Nevertheless most repetitive sequences do certainly neither contribute to the phenotype nor to the behaviour of the organisms. In consequence most of the repetitive DNA sequences have been regarded as ‘junk and jumble’ of the evolutionary past in the respective species 131. Yet irrespective if these sequences should be categorized as ‘innocent’, ‘ignorant’, ‘illusive’, ‘selfish’ or ‘parasitic’ (for a discussion of this semantic subject see Refs. 4 and S), their major present-day meaning can be seen in their successful applications as handy tools for various diagnostic endeavours [6]. There are several types of repetitive elements, among which five pr~ominant classes emerged quite consistently in the eukaryote genomes studied intensively so far (for review see e.g. Ref. 7). Variable number of tandem repeats (VNTR) have in common that their basic unit is repeated several to many times in a head,-to-tail fashion, VNTR should be subclassified into: (A) Simple repeat sequences and [8] and (B) the more complex ~j~~sa~e~~i~es 19,101. Simple repeats consist of short sequence motifs up to 10 bases repeated over and over in a tandem orientation (the upper length limit of the motifs in this category is still discussed). The minisatellites have a longer periodicity due to the larger basic core sequence. They could well have developed from initially perfect simple repeats by sequence divergence during evolution, additional amplification and secondary redistribution. (C) Short interspersed ~uc~earideelements (‘SINES’) are less than 500 base pairs long. Eminent examples exist like the well-known Alu sequences in man and the evolutionarily related Bl elements in the mouse. These repeat elements are quantitatively prevalent in many animal (and plant) genomes with up to millions of copies distributed all over the chromosomal complement though not completely randbm (for comprehensive review and definitions see Ref. 11). (D) i,ong interspersed ~uc~ea~ideelements (‘LINES’) harbor several kilobases of length and show often characteristic signs of retroposons [12]. (E) Extremely long runs of simple repeats (see A) or their degenerated, sequence divergent spin-offs constitute classical satellite DNA, which can in most cases be separated physically from the bulk of the remaining genome by bouyant density gradient cent~fugation. Using cytogenetic staining methodolo~, this material can often be visualized as so-called constitutive heterochromatin (see Ref. 13). Large chunks of this particular type of DNA are sometimes accumulated on the sex
s7
chromosomes of many (animal) species in which location it apparently does neither harm nor matter at all. Among these DNA sequences in man the cu (alphoid) and /3 satellites have been characterized in extenso [ 141. Also in this alphoid DNA a core motif of about 171 base pairs in length could be identified. Until now the full functional or biological meaning of most untranscribed and untranslated repetitive DNA sequences remained completely obscure. Nevertheless repetitive DNA has been employed as versatile tools in many areas where molecular biology techniques are being utilized. The following statements represent a listing of various diagnostic fields, where DNA probes containing repetitive sequence elements are currently already being applied successfully. Individuality Testing including Analyses in the Forensic Sciences Tools
A4ultilocus probes such as hypervariable minisatellites [9, lo], Ml 3 phage derived sequences (151, simple repetitive sequences [6,16,17] are freely available (though patented for commercial purposes) and sufficiently well characterized in the human and many animal and plant genomes. Probes are either molecularly cloned or synthetic oligonucleotides. in addition cloned and/or synthetic oligonucleotide probes specific for individual hypervariable loci have been developed which contain (either repetitive or more often) single copy sequences (allele specific oligonucleotides ‘ASOs’; for a compilation see Ref. 18). These markers are usually investigated and demonstrated by blot or direct in-gel hybridization. The latter procedure avoids the necessity for time-consuming blotting. In the last several months a wealth of highly informative simple repeal polymorphisms (SRPs; sometimes called ‘microsatellites’) has been published (as of today close to 300). Many more are available in individual laboratories. These DNA loci contain mostly (ca),/(gt), or related short simple sequences. They are localized in the genomic spacer regions (between genes), in 5 ‘-and 3 ‘-untranslated parts of messages as well as in introns of genes. Already all human chromosomes are covered in close to sufficient ‘density’ to map the complete human genome physically by SRPs. (Though human Y chromosomal loci have not yet appeared in press, we have characterized several of them in the Y-specific and the pseudoautosomal regions [19]). The major advantage of SRPs concerns the extreme degree of informativity (polymorphism information content, PIC) and an exactly definable chromosomal localization of these mulrial~elic markers as well as the speed with which they can be generated from chromosome-specific or ordered cosmid libraries and applied to all kinds of studies based on the linkage concept. For many of the SRPs, data on the somatic and meiotic mutation rates, etc. have yet to be established. Techniques
Multilocus fingerprints can be produced on Southern-blotted DNA samples (see above [9,10]) or preferably directly in situ in the gel [2U]. In general single copy DNA loci are probed from conventional agarose gels (mostly blotted onto supporting
membranes) or after amplification by polymerase chain reaction (PCR) of individual hypervariable loci on agarose or preferably on high resolution polyacrylamide or even denaturing sequencing gels. The abovementioned locus-specific SRPs are amplified by PCR and they can also be analyzed gelelectrophoretically. Applications
(a) Typing/matching for transplantation purposes (bone marrow, kidney, heart, liver, lung, etc.), zygosity determination in twins, detection of somatic mosaicism and chimerism, monitoring of graft-versus host reactions after blood transfusions (see e.g. Refs. 21 and 22); (b) animal and plant species and/or individual identification [6,9- 10,17,23], including mixing of blood samples, burnt bodies, parts of bodies [24); (c) paternity and/or family relationships, also possible in incest and deficiency cases [9,10]; (d) identification of stains [25J; (e) identification of cultured cells [26,27]; (f) studies in the behavioural sciences [27a,b]. Advantages
in comparison
to conventional
and other methods
For routine individualization cases (e.g, paternity), DNA fingerprinting using multilocus probes represents a quick, cheap and technically comparatively simple technique which is several orders of magnitude more informative with respect to individualization than other approaches (bloodgroups and other serology, HLA typing, obsolete phenotypic trait comparison procedures). One single analysis is sufftcient as compared to the multiple different experimental steps involved for determining the polymorphisms of serum enzymes etc. In the meantime the formerly disputed statistical background for the probability calculation of paternity with multilocus probes has largely been settled [28-301 and also cases with mutations in the offspring and incest as well as deficiency cases (incomplete trios) can be treated with the appropriate statistics. If necessary the sensitivity in stain analysis can be scaled up to the theoretical maximum by PCR, i.e. to demonstrate one single molecule (cave the ubiquitous danger of contaminations, which is intrinsic to the technique of the PCR but never discussed and appreciated appropriately). The simultaneous PCR of 3-5 highly informative SRP loci renders sufficiently differentiating individualization probabilities in most instances [ 191. Determhation
of Sex
in MM,
Animals ad Plants
Tools
Preferably Y chromosomal repeats are used, e.g. the Ha&I-produced 2.1 or the 3.4 kilobase repeat sequences. It is also possible to employ single copy sequences though naturally with reduced sensitivity [31,32]. Also birds (reptiles, fish and plants?) can be sexed by repetitive W- or Y-chromosomal sequences present only in the females (ZUZW sex chromosome mechanism) or the males (XXXY system).
s9
Techniques Direct hybridization to Southern-blotted, genomic DNA or PCR amplified material is most often appropriate 1331. It is even possible to start from maternal blood since fetal cells cross the placenta (see e.g. for single copy sequences [34]) Previous pregnancies with male fetuses have to be accounted for since lymph~ytes may last for years undetected by the immune system in the maternal circulation. {a) Demonstration of Y chromosomal DNA from blood, all other tissues and stains; (b) prenatal diagnosis for X-chromosomally inherited diseases; (c) preimplantation screening with single cells of the embryo; (d) sperm diagnosis; (e) animal and plant breeding (see below). Advantages in comparison fo other melhods This is a fast and simpk procedure since cell culture is not necessary. Theoretically the PCR is even possible from single cells obtained, e.g. from preimplantation em-. bryos or from blood of the pregnant mother (cave: ~ontaminat~ons~ see above and below). Diagnosis of Infectious Agents (Eukaryotic Organisms like Ciliates, Fungi, Protozoa, e.g. LRiskmaniaad Trypanosoma Species)
Simple repetitive ohgonucleotide
sequences.
Techniques Preferably by in-gel hybridization, Southern blot hybridizations are less advisable. {a) Species/strain identification; (b) differentiation of pathogenetic versus nonpathenogenetic strains of one species, e.g. in fungi and ciliates; (c) monitoring of genomic changes in the genomes of the infectious agents during the course of the
disease/infection. Advantagescompared to other methods The fast and simple (sub-) classification procedure is possible without extensive culture of the agents and without any laborious biochemical analysis of the organisms (if at all possible using non-fingerprinting methods). For several of the parasites biochemical marker analyses ,have not even been described. Yet even pathotyping in fungi has already been established [35]. Tumors Tools
The whole collection of different simple repetitive ohgonucleotide
sequences can
be used efficiently for a genera1 ‘genome scanning’ approach. Since the minisatellites and Ml3 target sequences are preferentiaily Iocalized only on the telomeres, they are sometimes less informative. Techniques See Individuality testing.. .. (a) Determination of clouality and of the relationship of primary tumors to metastases; verification of secondary independent tumor development; (b) screening for DNA amplification events; (c) changes in the DNAmethylation status; (d) monitoring of chromosomal losses and rearrangements; (e) and other chromosomal aberrations (duplications, partial deletions, mono/trisomies, etc.). Advantages compared to other methods Using approximately ten different oligonucleotide probes this sensitive, simultaneous and simple detection method achieves the analysis of many scattered DNA loci across the whole genome (the whole chromosomal complement). In Situ Hybridi&on/Chromo
Pninting in Man and Anh~ls
Tools
See Individuality resting . ... In addition different classical satellite sequences are useful for particular purposes. Techniques See Individuality testing .. . . The target DNA is detected directly on microscopic slides, preferably with non-radioactive reporter groups such as fluorochromes, biotin, digoxigenin or directly to the probe coupled alkaline phosphatase. The last version of probes is less advisable because of possible steric hindrances during hybridization. (a) Localization of individual repeat sequences on metaphase chromosomes; (b) identification of centromeres by alphoid satelIite sequences; (c) identification of nucleolus organizer regions (NORs) by 6 satellites or interspersed simple repeats; (d) repeat sequence ‘architecture’ in interphase nuclei; (e) histologic preparations. Advantages in comparison to other methods These include the higher sensitivity as compared to single locus probes due to the repetitive nature in a given locus and/or many hybridization events at different sites. Therefore also non-radioactive approaches are possible which are faster (signal developing time) and more precise in the subcellular microscopic localization (no range of the signal like with e-irradiation, e.g. from 32P-, “S-label or from [3N]thymidine). Most importantly no special precautions for radioactivity are necessary.
Animal (nnd plant) Ikediug
See individuality testing . ... Different fields of applications are open for the different multilocus probes (minisatellites; Ml3 sequences and simple repeats). As of today the largest spectrum of species (> 200 fungi, plants and animals} seems accessible by using simple repetitive oligonucleotide probes. Techniques See Individualify testing . .. . (a) De termination of paternity, maternity, zygosity and other genetic relationships for behavioural or socio-biological and experimental animal studies; (b) meat and animal identification (also for forensic purposes); (c) delineation of quantitative traits in breeding experiments; (d} efficient .inbreeding control for farm and/or laboratory animals; (e) determination of genetic relationships in endangered species (e.g. zoo or wild life animals) for selective breeding in order to maintain the gene pools as large as possible and therefore maximum heterozygosity; (f) identi~cation of the sex in species, where it is not possible without life endangering methods, like endoscopy for birds; (g) identification of cultured cells and contaminations therein. Advantages
compared to other techniques
DNA fingerprinting is highly informative for individualization. It is performed rapidly and technically simple and in addition also comparatively cheap. One principal technique is sufftcient for all aforementioned applications. Depending on the probe more or less the whole genome can be covered with interspersed simple repeat loci. Enformative simple repetitive oligonucleotide probes are available for all species tested so far. Genome Mapping and Aaalytis Tools
In order to characterize and to map large (parts of) eukaryotic genomes recently several simple repetitive nucleotide repeat sequences have been employed (mainly dinucleotide SRPs like (ca}J(gt)n or (ct)J(ga),; see above under Individuality testing ._.). The longer the perfect simple repeat stretches are, the more variable and hence informative are these DNA loci. Upon flanking DNA sequence analysis two antiparallely orientated primers are synthesized on both sides of the repeat and the standard PCR reaction is performed. Techniques
Amplification by PCR and gel electrophoretic analysis by staining or probe hybridization according to conventional protocols. Cave contaminations, especially
when the amount of the sample analyzed is very small (only few molecules from stains); therefore many appropriately designed contamination controls have to be performed routinely. Applications The procedure allows the ordering of genomic phage, cosmid and yeast artificial chromosome (YAC) libraries into long physically coherent DNA (chromosome) segments, the so-called contigs. Large scale genomic mapping projects. Linkage analysis to (disease) genes. Advantages in comparison to other and conventional methods The high informativity in comparison to conventional restriction fragment length polymorphisms (RFLPs) renders it a rapid and eficient analysis of many samples in very short time. In addition the number of necessary investigations (informative families) can be reduced considerably in order to firmly establish e.g. genetic linkage of a marker locus to a disease gene. References 1 Hinegardner R. Evolution of genomt size. In: AyaJa FJ. ed. Molecular evolution. Sunderland: Sinauer, 1976;179- 199. 2 L&bold DM, Swergold GD, Singer MF, Thayer RE, Dombroski BA. Fanning TG. Translation of LINE-DNA elements in vitro and in human cells. Proc Nat1 Acad Sci USA l!I90;87:6990-6994. 3 Uhno S. So much junk in our genome. Brookhaven Symp Biol 1972;23:366-370. 4 ‘Mobile DNA’. in: Berg and Howe, ed., Wiley and Sons, 1989. 5 Epplen JT. On simple repeated GATAIGACA sequences in animal genomes: A critical reappraisal. 3 Hered 1988;79:409-417. 6 Epplen JT, Ammer H, Epplen C et al. Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes. In: Burke G, Dolf G, Jeffreys AJ. Wolff R. eds. DNA fingerprinting: Approaches and applications. Basel: Birkhiiuser, ~99150-69. 7 Studer R, Epplen JT. On the organization of animal genomes; ubiquitous interspersion ofrepetitive DNA sequences. in: Geldermann H. Ellendorff F, eds. Genome analysis in domestic animals. Wcinheim/New York: VCH-Veriag, 1990;17-33. 8 Tautz D, Rent M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Rcs 1984;12:4f 27-4 I 38. 9 Jeffreys AJ, Wilson V, Thein SL. Hypervariable “minisatellite” regions in human DNA. Nature 1985a;314:67-73. 10 Jeffrcys AJ, Wilson V, Thein SL. Individual-specific fingerprints of human DNA. Nature 1985b;316:76-79. 11 Singer MF. SINES and LINES: Highly repeated short and long interspersed sequences in mammalian genomes. Cell 1982;28:433-434. 12 Rogers J. Retroposons defined. Nature 1983;301:460. 13 Advanced techniques in chromosome research. Adolph KW, ed. New York: Marcel Dekker 1991;1-462. 14 Waye JS, Willard HF. Human #3satellite DNA: genomic organization and sequence definition of a class of highly repetitive tandem DNA. Proc Natl Acad Sci USA 1989$6:6250-6254. IS Vassart G, Georges M. Monsieur R. Brocas H, Lequarre AS, Christophe D. .4 sequence in M I3 phage detects hypervariable minisatellites in human and animal DNA. Science 1987;235:683-684. 16 Ali S. Miiller CR, Eppien JT. DNA fingerprinting by oligonucleotides specific for simple repeats. Hum Genet 1986;74:239-243.
s13 17 Weising K, Ramser J, Kaemmer D, Kahl G, Epplen JT. Oligonucleotide fingerprinting in plants and fungi. In: Burke T, Dolf G, Jeffreys AJ, Wolff R, eds. DNA fingerprinting: Approaches and applications. Basel: Birkhiiuser, 1991;312-329. 18 Human Gene Mapping Library, Yale University/Howard Hughes Medical Institute, New Haven, Connecticut. 19 Roewer L, Arnemann J, Spurr NK, Grzeschik K-H, Epplen JT. Simple repat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts. Hum Genet in press. 20 Tsao SGS, Brunk CF, Pearlman RE. Hybridization of nucleic acids directly in agarose gels. Anal Biochem 1983;131:365-372. 21 Ugozzoli et al. Genotypic analysis of engraftment in thalessemia following bone marrow transplantation using synthetic oligonucleotides. Bone Marrow Transpl 1989;4:I73- 180. 22 Kunstmann E, Backer T, Sauer H, Mempel W, Epplen JT. Diagnosis of transfusion-associated graft-versus-host disease by genetic fingerprinting and polymerase chain reaction. Transfusion, in press. 23 Niirnberg P, Roewer L, Neitzel H et al. DNA fingerprinting with the oligonucleotide probe (CAC)s/(GTG),: somatic stability and germline mutations. Hum Genet 1989;84:75-78. 24 Piiche H, Wrobel G, Schneider V, Epplen JT. The identification of a charred body by oligonucleotide fingerprinting with the (GTG)5 probe. In: Berghaus G, Brinkmann B, Rittner C, Staak M, eds. DNA-technology and its forensic application. Heidelberg: Springer-Verlag, 1991;192-195. 2.5 Roewer L, Niirnberg,P, Fuhrmann E, Rose M, Prokop 0, Epplen JT. Stain analysis using oligonucleotide probes specific for simple repetitive DNA sequences. Forensic Sci Int 1990,47:59-70. 26 Gilbert DA, Reid YA, Gail MH et al. Appli~tion of DNA ~ngerp~nts for cell-line individualization. Am J Hum Genet 1990,47:499-514. 27 Speth C, Epplen JT, Oberbaumer I. DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor. In Vitro Cell Dev Biol 1991;27A:646-650. 27a Burke T, Davies NB, Bruford MW, Hatchwell BJ. Parental care and mating behaviour of polyandrous dunnocks Pruneita modularis related to paternity by DNA fingerprinting. Nature 1989;338:249-251. 27b Achmann R, Heller K-G, Epplen JT. Last male sperm precedence in the bushcricket Poecittimon vetuchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting. Mol Ecol 1922;in press. 28 Evett IW, Werrett DJ, Buckleton JS. Paternity calculations from DNA multilocus profiles. J Forensic Sci Sot 1989;29:249-254. 29 Yassoilridis A, Epplen JT. On paternity determination from multilocus DNA profiles. Electrophoresis 1993;12:221-226. 30 Krawczak M, Biihm I, Nib&erg P, et al. Paternity testing with probe (GTG)~/(CAC)~: a multicenter study. Hum Genet 1992; in press. 31 Page DC, Mosher R, Simpson EM et al. The sex-determining region of the human Y chromosome encodes a finger protein. Cell I987;51:1091-1104. 32 Sinclair AH, Berta P, Palmer MS et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990;346:240-244, 33 Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768-770. 34 Ebensperger C, Studer A, Epplen JT. Specific amplification of the ZFY gene to screen sex in man. Hum Genet 1989;82:289-290. 35 Weising K, Kaemmer D, Epplen JT, Weigand F, Saxena M, Kahl G. DNA fingerprinting of Ascochylu rabiei with synthetic oligodeoxynucleotides. Curr Genet 1991;19:483-489.
