DNA Profiling and DNA Fingerprinting, 1st Edition

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Editors:

Prof. Dr. JorgT. Epplen Dr. Thomas Lubjuhn

Molekulare Humangenetik Institut fUr Evolutionsbiologie und Dkologie
Ruhr-Universitat Bochum An der Immenburg 1
44780 Bochum 53121 Bonn
Germany Germany

Library of Congress Cataloging-in-Publication Data

DNA profiling and DNA fingerprinting / edited by Jorg T. Epplen, Thomas Lubjuhn
p. cm. - - (Methods and tools in biosciences and medicine)
Includes bibliographical references and index.

1. DNA fingerprinting. I. Epplen, Jorg T., 1952-

II. Lubjuhn, Thomas, 1962- III. Series.
QP624.5.D72D65 1999
610'.28- -dc21 99-10116
CIP

Deutsche Bibliothek Cataloging-in-Publication Data

DNA profiling and DNA fmgerprinting/ ed. by Jorg T. Epplen; Thomas Lubjuhn.-
Basel; Boston; Berlin: Birkhauser, 1999
(Methods and tools in biosciences and medicine)

ISBN 978-3-7643-6018-4 ISBN 978-3-0348-7582-0 (eBook)

DOI 10.1007/978-3-0348-7582-0

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 v

Contents

List of Contributors _._..._ _ _._..

__._._----------
VII
Preface ---_.__.._._._.._._. IX
Abbreviations ____.__....._._.___...._..______. X

1. DNA Fingerprinting of Prokaryotic Genomes _ _ _ _ _ _ _ __ 1

Paul D. van HeIden
2. Plant DNA Fingerprinting and Profiling 17
Pierre Saumitou-Laprade, Yves Piquot, Olivier Raspe,
Jacqueline Bernard and Klaas Vrieling
3. DNA Fingerprinting and Profiling in Behavioural
Ecology _ __ 39
Thomas Lubjuhn and Klaus Peter Sauer
4. DNA Profiling in Veterinary Medicine _ _ _ _ _ _ _ _ _ __ 53
Johannes Buitkamp, Rudi Antes and Volker Wagner
5. Multilocus DNA Fingerprinting _ _ _ _ _ _ _ _ _ _ _ __ 71
Jorg Schmidtke
6. Various Levels of (Epi)Genetic Diversities as
Demonstrable via Simple Repeated Sequences _ _ _ _ _ _ __ 83
Jorg T. Epplen, Cornelia Epplen and Winfried Maueler
7. Forensics: Analysis of Short Tandem Repeat Loci by
Multiplex PCR and Real Time Fluorescence Detection
During Capillary Electrophoresis _ _ _ _ _ _ _ _ _ _ _ __ 101
Bruce Budowle and Tamyra R. Moretti
8. Mitochondrial DNA: Diversity Analysis and Possible
Pitfalls _.__._____________ 117
Hans Zischler
9. PCR-based DNA Profiling of Human Y Chromosomes 133
Fabricio R. Santos, Denise R. Carvalho-Silva and Sergio D.J. Pena
10. The Use ofImperfect Microsatellites for DNA
Fingerprinting and Population Genetics 153
Christian Schlotterer and Barbara Zangerl
11. PCR-SSCP: A Method for Reliably Detecting Single
Nucleotide Polymorphisms in Multifactorial Diseases 167
Silke Jackel, Jorg T. Epplen and Cornelia Epplen
VI

12. Amplified Fragment Length Polymorphisms:

A Non-Random peR-Based Technique for Multilocus
Sampling 177
Timothy F. Sharbel
13. Two-Dimensional DNA Fingerprinting _ _ _ _ _ _ _ _ _ __ 195
Karola Marczinek, Jochen Hampe and Peter Niirnberg
14. Statistical Approaches and Methods in Population
Genetics Using Microsatellite Data .'_ _ _ _ _ _ _ _ _ _ __ 215
Eduardo Jose Melo dos Santos
15. Statistical Inference from DNA Evidence _ _ _ _ _ _ _ _ __ 229
Michael Krawczak

Guide to Solutions ___. 245

Guide to Protocols 246
Troubleshooting Guide 248
Index _ _ _, 249
 VII

List of Contributors

RUDI ANTES, Lehrstuhl fUr Tierzucht, Technische UniversWit Munchen,

D-85350 Freising-Weihenstephan, Germany
JACQUELINE BERNARD, Laboratory of Genetics and Evolution of Plant Popula-
tions, URA CNRS 1185, Scientific and Technical University of Lille, SN2,
59655 Villeneuve d'Ascq CEDEX, France
BRUCE BUDOWLE, Forensic Science Research, and Training Center, FBI Acad-
emy, Quantico, Virginia 22135, USA
JOHANNES BUITKAMP, Lehrstuhl fUr Tierzucht, Technische UniversiHit Munch-
en, D-85350 Freising-Weihenstephan, Germany
DENISE R. CARVALHO-SILVA, Departamento de Bioquimica, Instituto de CH~ncias
Biologicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 30161-
970 Belo Horizonte, Brazil
CORNELIA EpPLEN, Molekulare Humangenetik, Ruhr-UniversWit Bochum,
44780 Bochum, Germany
JORG T. EpPLEN, Molekulare Humangenetik, Ruhr-UniversWit Bochum, 44780
Bochum, Germany
JOCHEN HAMPE I. Medizinische Klinik, Christian-Albrechts-UniversWit,
SchlittenhelmstraBe 12, D-24105 Kiel, Germany
PAUL D. VAN HELDEN, MRC Centre for Molecular & Cellular Biology, Dept. of
Medical Biochemistry, University of Stellenbosch, P.O. Box 19063, Tyger-
berg, 7505, South Africa
SILKE JACKEL, Molecular Human Genetics, Ruhr-University, 44780 Bochum,
Germany
MICHAEL KRAWCZAK, Institute of Medical Genetics, University of Wales College
of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom
THOMAS LUBJUHN, Institut fUr Evolutionsbiologie und 0kologie, An der Immen-
burg 1, 53121 Bonn, Germany
WINFRIED MAUELER, Molecular Human Genetics, Ruhr-University, 44780
Bochum, Germany
KAROLA MARCZINEK, Institut fUr Medizinische Genetik, Universitatsklinikum
Charite, D-10098 Berlin, Germany
TAMYRA R. MORETTI, Forensic Science Research and Training Center, FBI
Academy, Quantico, Virginia 22135, USA
PETER NURNBERG, Institut fUr Medizinische Genetik, Universitatsklinikum
Charite, D-10098 Berlin, Germany
SERGIO D.J. PENA, Departamento de Bioquimica, Instituto de Ciencias Biologi-
cas, Universidade Federal de Minas Gerais, Caixa Postal 486, 30161-970
Belo Horizonte, Brazil
YVES PIQUOT, Laboratory of Genetics and Evolution of Plant Populations, URA
CNRS 1185, Scientific and Technical University of Lille, SN2, 59655 Ville-
neuve d'Ascq CEDEX, France
VIII

OLIVIER RASPE, Unity of Ecology, Catholic University of Louvain, 5 Place Croix-

du-Sud, 1348 Louvain-Ia-Neuve, Belgium
EDUARDO JosE MELO DOS SANTOS, Laboratorio de Genetica Humana e Medica,
Universidade Federal do Para, Caixa Postal 8615, 66075-970 Belem, Para,
Brazil
FABRICIO R. SANTOS, Departamento de Biologia Geral, Instituto de Ciencias
Biologicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 30161-
970 Belo Horizonte, Brazil
KLAUS PETER SAUER, Institut fUr Evolutionsbiologie und 0kologie, Rheinische
Friedrich-Wilhelms-UniversWit Bonn, An der Immenburg 1, 53121 Bonn,
Germany
PIERRE SAUMITOU-LAPRADE, Laboratory of Genetics and Evolution of Plant
Populations, URA CNRS 1185, Scientific and Technical University of Lille,
Bat. SN2, 59655 Villeneuve d'Ascq CEDEX, France
CHRISTIAN SCHLOTTERER, Institut fUr Tierzucht und Genetik, Josef-Baumann
Gasse 1, 1210 Wien, Austria
JORG SCHMIDTKE, Institute of Human Genetics, Hannover Medical School, Carl-
Neuberg-Str. 1, D-30625 Hannover, Germany
TIMOTHY F. SHARBEL, Arbeitsgruppe Michiels, MPI fUr Verhaltensphysiologie
Seewiesen, Postfach 1564, D-82305 Starnberg, Germany
KLAAS VRIELING, Institute of Evolutionary and Ecological Sciences, Van der
Klaauw Laboratory, PO Box 9516,2300 RA Leiden, The Netherlands
VOLKER WAGNER, Genedia® Molekulargenetische Begutachtung GmbH, Theo-
dolindenstr. 97, D-81545 Miinchen, Germany
BARBARA ZANGERL, Institut fUr Tierzucht und Genetik, Josef-Baumann Gasse 1,
1210 Wien, Austria
HANS ZISCHLER, AG Primatengenetik, Deutsches Primatenzentrum GmbH,
Kellnerweg 4, D-370n Gottingen, Germany
 IX

Preface

This book reflects practical approaches to answering a variety of biological

and medical questions using DNA fingerprinting and genetic profiling in a
broad sense that also includes statistical evaluation of the data. It has been
written for biology, biochemistry and medical students, graduates, postdoctor-
al fellows, academic teachers and for all researchers in the theoretical and ap-
plied sciences wherever genetic identification and relationship analyses are re-
quired. Although the book represents a holistic treatment of the "state-of-the-
art" technology concerning these topics, methological details are always given
in the form of elaborated protocols so that the reader can benefit in terms of
everyday work in the laboratoryas well.

Jorg Thomas Epplen Thomas Lubjuhn

Bochum, Germany Bonn, Germany

January 1999
x

Abbreviations
10 one-dimensional numtDNA nuclear-localized mitochondrial
2D two-dimensional DNA
A260 absorption at 260 nm origin of H-strand replication
A280 absorption at 280 nm origin of L-strand replication
AFLP amplified fragment length poly- polyacrylamide
morphism polyacrylamide gel electrophor-
AP-PCR arbitrarily primed PCR esis
BSA bovine serum albumin PAR pseUdo-autosomal region
CE capillary electrophoresis PBL peripheral blood lymphocyte
CNS central nervous system PCR polymerase chain reaction
CaDIS combined DNA indexing system PFGE pulse field gel electrophoresis
CpDNA chloroplastic DNA Phe phenylalanine
CPU central processing unit PP polypropylene
CTAB hexadecyltrimethyl ammonium PPVP polyvinyl polypyrrolidone
bromide Pro proline
DEAE diethylaminoethyl PVP polyvinylpyrrolidone
DGGE denaturing gradient gel electro- QTL quantitative trait locus
phoresis RAPD random amplified polymorphic
DHPLC denaturing high-pressure liquid DNA
chromatography RFLP restriction fragment length poly-
DIG digoxigenin morphism
D-Ioop displacement loop RNA ribonucleic acid
DTAB dodecyltrimethyl ammonium bro- rRNA ribosomal RNA
mide RT room temperature
DTT l,4-dithiothreitol SDS sodium dodecyl sulfate
ECL enhanced chemiluminescence SINE short interspersed nuclear ele-
EDTA ethylenediaminetetraacetic acid ment
EtBr ethidium bromide SMM stepwise mutation model
FISH fluorescence insitu hybridisation SNP single nucleotide polymorphism
GBA genetic bit analysis SSC standard saline citrate
HLA human leukocyte antigen SSCP single-strand conformational
HMW high molecular weight DNA polymorphism
HSP heavy-strand promoter sso sequence-specific oligonucleotide
H-strand heavy strand typing
HVR hypervariable region STR short tandem repeat
ICAM intercellular adhesion molecule TAE TrisiacetatelEDTA buffer
IFN interferon TAS termination -associated sequence
IL interleukin TBE TrisiboratelEDTA buffer
IS insertion sequence (element) TE Tris/EDTA buffer
LINE long interspersed nuclear ele- TEMED N,N ,N',N'-tetramethylethylene-
ment diamine
LSP light-strand promoter Tm melting temperature
LSSP low-stringency single primer TNF tumor necrosis factor
LST lymphocyte-specific transcript TNFR TNF receptor
L-strand light strand Tris tris (hydroxymethyl)-amino-
LT lymphotoxin methane
LTR long terminal repeat tRNA transfer RNA
MHC major histocompatibility complex UF denaturant consisting of 7 M urea
MS multiple sclerosis and 40% formamide
mtDNA mitochondrial DNA uv ultraviolet radiation
MVR-PCR minisatellite variant repeat PCR YAP Y Alu polymorphism
ND NADH dehydrogenase
Ne effective population size
11 DNA Fingerprinting of Prokaryotic
Genomes
Paul D. van HeIden

Contents
1 Introduction....................................................................................... 1
2 Methods................................. ............................................................ 3
2.1 General methods................. ........................................................... 3
Preparation ofDNA........................................................................ 3
Protocol 1: Isolation of DNA from Mycobacterium tuberculosis
grown on solid medium as an example........................................... 4
Protocol 2: Preparation of DNA.................................................. .... 5
Choice of probes for hybridization .................................................... 6
Profiles and multilocus patterns generated by PCR ............................. 7
2.2 Other methods........................................................ .. ..................... 9
3 Results and Discussion.......... .... .... .................. .... .... .... ..... .......... ..... ..... 9
3.1 Data interpretation and analysis...................................................... 9
3.2 Applications ...... ..... .... ............. .... .... ..... .... ........ ..... ...... ..... .... ..... .... 11
4 Troubleshooting.... .. ........................................................................... 12
Acknowledgments...................... .............................................................. 12
References...................................................................................... ........ 13

1 Introduction

DNA fingerprinting has become such an established technology that it has fea-
tured frequently in the media and is quite well known to the general public.
This is largely a result of publicity surrounding a small number of high-profile
criminal or litiginous cases. What is perhaps less well known is that the tech-
nology can also be applied to the study of prokaryotic organisms, where it has
many uses. These uses may be either simple or complex, and the exact technol-
ogy to be used will depend largely on the question to be asked or the hypothesis
to be tested. Some of the simple uses for genotyping of prokaryotes in the la-
boratory would be to test for contamination of cultures or alterations in strain
genotype during culture or manipulation. Further uses may concern the ecol-
ogy or taxonomy of prokaryotic organisms, or applications to agricultural, ve-
terinary or medical problems. In the latter cases, it may be to determine
whether a patient (animal or human) is afflicted by a mixed or single infection
of any given pathogen. Many additional questions can be asked concerning the
disease under investigation. Specifically, routes of transmission and index
cases can be sought, outbreaks can be investigated and disease dynamics fol-

Methods and Tools in Biosciences and Medicine

DNA Profiling and DNA Fingerprinting, ed. by J. T Epplen and T. Lubjuhn
© 1999 Birkhiiuser Verlag BaseVSwitzerland
2 Paul D. van HeIden

lowed in a broader context. These last few are examples of the new science of
molecular epidemiology, a marriage between traditional epidemiology and mo-
lecular biology.
A number of techniques have been developed for genotyping prokaryotes.
The particular technique to be used will depend on the information available
on the individual species being studied. As for eukaryotes, it is useful to identify
multiple loci for informative DNA fingerprints. Chromosomal regions contain-
ing repetitive elements are useful targets [1], as are insertion sequences [2-41,
usually present in multiple copies. Amongst the multilocus fingerprinting tech-
niques used will be simple restriction enzyme digests followed by visualization
on a gel [5] (this may be either simple restriction enzyme digests or pulse field
gel electrophoresis - PFGE [6]). However, these techniques may be less discri-
minating than others, since the fragments are so large that many polymorph-
isms will not be detected. These techniques will therefore not be discussed
further. Alternatively, a restriction enzyme digest may be followed by Southern
blot analysis. In this case the probes may be specific elements (genes) which
are known to be polymorphic, or be insertion elements (IS) or ribosomal probes
(ribotyping [7-9]). Polymerase chain reaction (PCR) technology may also be
used, either for random amplification (RAPD [10-14]) or amplification of speci-
fic loci known to be polymorphic [1, 15-17]. Finally, DNA sequencing may be
done or oligonucleotides [18-21] used to probe for polymorphisms in the gen-
ome by hybridization. Oligonucleotides may be used either to probe a mem-
brane with genomic DNA or alternatively may be fixed to a membrane in an ar-
ray and then labelled genomic DNA hybridised to the array of oligonucleotides
(spoligoblotting [4,22,23]).
The method used to examine an outbreak of a pathogen in a single hospital
ward, or in an enclosed domestic herd of animals, might be quite different from
that used to examine the disease dynamics of a pathogen within the borders of
a country or globally. In the case of a countrywide study for example, it will be
likely that a large number of organisms (cultures) will have to be examined,
which will generate a large number of fingerprints. A downline system will be
required to integrate and compare the data generated, as simple eye-hand
matching will not be possible. Furthermore, there is a question of accuracy to
be considered, i.e. how much tolerance (experimental or lab variation) will be
allowed? These considerations are not necessarily relevant if only 20 or fewer
isolates from a simple outbreak or laboratory investigation are concerned.
Some of the problems encountered in genotyping prokaryotes relate to their
relatively short generation time, and thus genome stability must be considered.
Certain areas of the genome may be quite invariant (stable), whereas others
may be hypervariable. The hypervariable locus may not follow a lineage (evo-
lutionary line) and is thus unsuitable for genotype analysis. For each study,
one needs to consider carefully the definition of a strain, clone or clonal var-
iant. A small genome change may make a large phenotypic difference, e.g. a
single point mutation on the chromosome leading to drug resistance. It is ex-
tremely unlikely that a change such as this will be detected by multilocus fin-
DNA Fingerprinting of Prokaryotic Genomes 3

gerprinting methods. Alternatively, a phenotypic change can be mediated by

epigenetic elements (e.g. plasmids [9]) and also not detected by fingerprint
analysis. The typing of strains by plasmid analysis will not be discussed here.
Clearly, chromosomally identical bacterial isolates may harbour identical or
different plasmids, and the same holds true for nonidentical bacterial isolates.
Equally, a DNA fingerprint change may not necessarily relate to a change of
phenotype.
This chapter outlines some of the techniques currently being used to study
prokaryotes and discusses some of their advantages and disadvantages, as well
as suggestions on the situation where each may best be used.

2 Methods

2.1 General methods

Genotype analysis of prokaryotes requires three steps: (1) DNA isolation (2)
generation of a DNA fingerprint using one or more methods and (3) analysis of
the resultant DNA fingerprints or profiles. There are many variations in all
three steps, and the following sections will provide some of the options avail-
able.

Preparation of DNA
The quality of the genomic fingerprint depends largely on the quality of DNA
used. The isolation of genomic DNA from bacteria depends largely on the nat-
ure of the cell wall of each species. A complex and tough cell wall (e.g. Klebsiel-
la, Mycobacterium sp.) complicates purification in comparison to that of bac-
teria with relatively fragile walls (e.g. Helicobacter pylori, Escherichia coli).
Some bacteria have cell walls with copious amounts of mucopolysaccharides,
which can adversely influence subsequent manipulation of DNA. The isolation
methods suggested should be modified according to the nature of the bacterial
cell wall (as suggested in the Notes to Protocol 1), as well as the source of the
bacterial culture, which may be harvested from solid or liquid medium.
4 Paul D. van HeIden

Protocol 1 Isolation of DNA from Mycobacterium tuberculosis grown on solid

medium [21] as an example
1. Use a Lowenstein-Jensen slant culture of M. tuberculosis which has visi-
ble colonies.
2. Heat at 80°C for 1 hour to kill bacteria (Note for pathogens).
3. Add 3 ml of extraction buffer (50 mM Tris-HC1, 25 mM EDTA, 5% mono-
sodium glutamate; pH 7.4) and carefully scrape the colonies off the slant,
using a disposable loop (estimated volume up to 100 Ill).
4. Pour the buffer and bacteria into a 50-ml polypropylene tube containing ±
30 5-mm-diameter glass balls.
5. Add another 3 ml of extraction buffer to the slant and remove remaining
colonies - pool into polypropylene tube.
6. Vortex the sample at full speed for 2 to 3 min to disrupt the bacterial colo-
nies. All clumps should be broken up.
7. Add 400 III of lysozyme stock (50 mg/ml H2 0) and incubate at 37°C for 2
hours Mix occasionally.
8. After 1.5 hours - add 10 III of RNase (10 mg/ml) to the sample and incu-
bate at 37°C for the remaining 30 min.
9. Add 600 III of 10 x proteinase K buffer (100 mM Tris, 50 mM EDTA, 5%
SDS; pH 7.8).
10. Add 150 III of proteinase K (10 mg/ml in H2 0).
11. Mix gently and incubate at 45 to 52°C for 16 hours PhenoVchloroform-ex-
tract and precipitate DNA with isopropanol. DNA can be "fished out" with
a glass rod if a large conglomerate is formed.
12. Alternatively, the mixture is left at -20°C for 30 min and then centrifuged
at 3000 rpm for 30 min.
13. Redissolve dry DNA (after ethanol wash) in TE (10 mM Tris-HC1, 1 mM
EDTA; pH 8.0).
Note:
For other bacteria, this protocol can be adapted as follows:
(1) Liquid cultures: inoculate 5 ml of medium with isolate. Grow to satura-
tion. Recover the bacterial pellet by microcentrifugation and resuspend in
approximately 35% original culture volume in TE. Add SDS and protei-
nase K (final concentrations 0.5% and 100 Ilg/ml, respectively). Lysozyme
and RNase A digestions may be done prior to addition of SDS and protei-
nase, if desired.
(2) Prior to the first phenol/CHCl3 extraction step, 5 M NaCI or 3 M NaAc
can be added, followed by addition of hexadecyltrimethyl ammonium bro-
mide (CTAB) to a final concentration of 10 to 12%. Mix and incubate for
10 min at 65°C. Proceed with extraction. (CTAB helps to remove polysac-
charides which can copurify with DNA, e.g. Klebsiella sp.).
DNA Fingerprinting of Prokaryotic Genomes 5

(3) It is desirable to be able to recover stringy precipitate with a rod, since

this represents high molecular weight, good-quality DNA. (4) If a PCR-
based method is to be used, it is possible in many instances to use rela-
tively crude source material, e.g. blood, urine, sputum [24, 25J. A partial
purification of the bacteria often improves results, as inhibitors of PCR
are present in many fluids, whether of plant or animal origins. This can
often be done simply by magnetic bead recovery or by centrifugation over
sucrose [26J. The bacteria may then be lysed by boiling and the solution
used directly as PCR substrate.

Protocol2 Preparation of DNA

Typically, 6 /lg of DNA may be incubated in 100 /ll of digestion buffer with 30
units of the restriction endonuclease at the recommended temperature for 2 to
16 hours. DNA can be concentrated by addition of 9 /ll of 3 M NaAC (pH 5.5), 9
/ll of 1 /lg//ll of transfer RNA (tRNA) and 330 /ll of 100% ethanol. The DNA re-
covered can be dissolved in 10 to 20 /ll of a loading buffer [6 x loading buffer is
30% glycerol, 0.25% w/v bromophenol blue, 0.6% SDS in TE (pH 8.0)].
Electrophoresis: Conditions for gel electrophoresis vary, but are typically as
follows: 0.8-1.2% agarose in TrislboratelEDTA buffer (TBE, pH 8.0), run over-
night in TBE at 2 V/cm, until the dye front is approximately 2 cm from the end
of the gel (size approximately 20 cm by 25 cm). Approximately 3 /lg of genomic
DNA per well is loaded (size of teeth in well-former, - 6 x 1.5 mm, or 35 Ill).
Shorter gels may be used, but longer gels enhance accuracy substantially. In
order to compare a large number of samples, attention must be given to repro-
ducibility and methods of comparison. Standards to measure fragment size
and to compensate for differences between gels are necessary. The use of ex-
ternal and internal standards is recommended. On a 20 lane-gel, external
standards of digested genomic DNA from a standard reference strain of the
species to be examined, or markers applied in lanes 1, 10 and 20, are useful.
For greater accuracy, internal standards can also be run. This should prefer-
ably be a ladder of markers covering the area spanned by all the detectable
elements that will not hybridize to the probe to be used. We have found Marker
X useful (Boehringer Mannheim, at 250 /lg/ml), added to a concentration of 6.6
/ll to 8 ml of 2 x loading buffer and added to the genomic DNA prior to electro-
phoresis.
Southern blotting: Capillary transfer [27] to Hybond N+ (Amersham) using 20
x SSPE (3 M NaCl, 0.2 M NaH zP0 4 , 20 mM EDTA; pH 7.4). Rinse membrane in
2 x standard saline citrate (SSC) (pH 7.0) and fix DNA to the membrane by bak-
ing at 80 DC for 2 hours. The membrane should be marked to aid the orienta-
tion of markers and test samples (e.g. corner "spots" of DNA, which will also
hybridize).
6 Paul D. van HeIden

Choice of probes for hybridization

RFLP analysis requires thought with respect to the combination of restriction
enzyme and probe to be used and will often require empirical testing, particu-
larly when the reader will be working with previously unresearched material
(see also chapter of Lubjuhn and Sauer, this volume). The choice of probes is
primarily oligonucleotides of regular repeat sequences, e.g. (GTGh, (GACA)4,
or alternatively insertion elements, ribosomal RNA (rRNA) gene sequences, re-
petitive elements or specific genes containing common polymorphisms. A sepa-
rate hybridization with marker DNA may be necessary, if this differs from test
DNA (e.g. internal markers).
Simple repeat oligonucleotides: The genomes of both eukaryotic and pro-
karyotic organisms contain regions where simple repeats, e.g. (GTGh, (GACA)4
occur [18-21, 28]. These areas frequently identify multiple polymorphic loci,
and thus simple repeat oligonucleotides (while not necessarily always finding
perfect matches) can be used as DNA fingerprinting probes, particularly where
little is known of the organism and its genomic sequence. Now that consider-
able genome sequences are available, it is possible to identify exactly where
and what simple repeats could be used. The key to this technique lies in finding
the correct enzyme and probe combination - in the absence of any other infor-
mation, this must be found empirically. The technique consists of hybridization
of a labelled oligonucleotide to a Southern blot membrane and visualization by
autoradiography or other means. These methods have been described in de-
tail, and follow standard membrane hybridization protocols [18-21]. An inter-
esting variation is to hybridize to the genome directly in an agarose gel, as
prior transfer of genomic DNA to a membrane is not necessarily required. In
this case, the agarose gel (not more than 1%, preferably) is first dried under va-
cuum, after which it is soaked in denaturant (0.5 M NaOH, 1.5 M NaCl) for 30
min at room temperature. This is followed by neutralization (0.5 M Tris-HCl,
pH 8.0) for 30 min at room temperature. The gel is then treated as for a mem-
brane, with prehybridization, hybridization and washing steps [21].
IS elements: Certain elements within the genome of prokaryotes are trans-
posable, namely transposons. Many different families of these elements have
been identified in various bacterial species, which can occur at variable copy
number (e.g. IS6110 in M. tuberculosis, which occurs at from 0 to 25 copies
per cell [2, 4]) and at variable locations. These elements can be particularly
useful, as they often provide a very clear, unequivocal multilocus pattern,
where each band is of equal intensity. Lineages are usually relatively stable.
These elements can also be used to generate a number of different DNA finger-
prints, since by using a restriction enzyme that cuts within the IS element, one
may use the 5' and 3' portion of the IS as different probes to test polymorph-
isms flanking the insertion element. By using a restriction enzyme not included
in the IS element, a third variant of the DNA fingerprint may potentially be ob-
tained.
DNA Fingerprinting of Prokaryotic Genomes 7

No knowledge of the insert sites is required in order to generate a finger-

print. One requires either a cloned IS element to use as a probe, or alterna-
tively one must make a labelled PCR probe using primers complementary to
the IS sequence and sited such that either the entire IS is used as a probe, or
the 5' or 3' side only. Typically, probes may be labelled for detection using the
ECL (enhanced chemiluminescence) system according to the manufacturer
protocols and hybridized according to standard procedures [2].
Polymorphic genes: A number of polymorphic genes have been identified in
bacteria which may be used as polymorphic probes to type strains [15, 16, 29].
The procedures utilize labelling and hybridization techniques as described
adequately elsewhere.
Repetitive elements: The genome of prokaryotes may contain highly re-
peated sequences at certain loci. These may be direct repeats of variable
lengths [1, 30] or repeats interspersed with unique sequences [4, 31]. The
number and type of repeats and interspersed regions may vary considerably
and thus can be a target for profile analysis. Probes may be an oligonucleotide
of the repeat, or alternatively a longer, cloned sequence from a strain of that
species. These probes may be labelled and used as described elsewhere. If the
repeat unit is quite short (e.g. 24 bp), then it is likely that small variations may
not be accurately detected by Southern blot analysis. Such polymorphisms are
usually more profitably analysed using PCR, as outlined later.
Ribotyping: Polymorphisms in the ribosomal operon of bacterial species
have been found and this operon can therefore be used as a probe for DNA fin-
gerprint or profile analysis [7-9]. Either a cloned DNA operon or ribosomal
RNA may be used as source material for hybridization. A cloned probe may be
labelled by random-prime labelling or for ECL detection. Alternatively, riboso-
mal RNA (16S and 23S) from the organism or another species (e.g. E. coli) can
be used as a template for the production of a labelled probe by random oligo-
priming using hexanucleotides (Pharmacia) and reverse transcriptase.

Profiles and multiloeus patterns generated by peR

PCR technology has revolutionized molecular biology and has had a substantial
impact on DNA profiling. There are essentially three different conceptual ap-
proaches for profiling prokaryotes. Directed (targeted to specific sequences),
semidirected or random. The advantages of using PCR for profiling are that the
method may be applied to crude samples and is extremely rapid. However,
there are some disadvantages (see below). PCR is such a well-known technol-
ogy that full details of every method will not be provided, but outlines and sui-
table references will be given.
Random approach: The random approach [10, 12, 14] characterized by
RAPD-PCR (or arbitrary primed - AP-PCR) is useful particularly in studying an
unknown genome. However, these methods have reproducibility problems [13,
32], and the large variation in signal intensity within a sample renders them
complex to analyse. Nevertheless, they have proved quite popular. Between 2
8 Paul D. van HeIden

and 40 random primers of 10 to 20 bases in length may be used in a single PCR

reaction containing 1-20 ng of DNA. The conditions used are frequently as fol-
lows: incubate the reaction mixture for 3 min at 94 to 95°C. Cycle for 40 re-
peats including denaturation for 30 sec at 94 °C, anneal for 60 sec at 36 to
40°C and extend for 1 min at 72 °C. PCR products may be visualized by ethi-
dium bromide staining. The annealing temperature will depend on the primers
used, and it may be possible to generate a more reproducible and specific pat-
tern using fewer primers and higher temperature. This technique is sensitive
to the quality of the template used, the accuracy of the thermocycler and con-
tents of the buffer.
These techniques are very useful for the identification of new polymorphic
loci in known and unknown genomes and useful for rapid prescreening of large
numbers of samples before a definitive analysis is done. The information con-
tent obtained is high, but the manipulation of the results may be complex and
difficult.
Semidirected PCR: This technology is characterized by prior digestion of the
genome with restriction enzymes followed by PCR. The methodology is dealt
with in detail elsewhere (see Sharbel, this volume). The technology may be ap-
plied to known or unknown genomes and consists of prior digestion of the gen-
ome with a restriction enzyme followed by addition of oligonucleotide adapters
to the fragments generated and PCR amplification of the fragments based upon
the oligonucleotide linkers. The information content of this method is high, and
unlike RAPD-PCR, it gives reproducible bands of more even intensity. The
method may be applied to testing a large number of variable genetic markers
(which will be unknown) or may be applied to specific sequences, such as in-
sertion elements [33]. In the latter case, one of the linkers, at least, will be com-
plementary to the insertion element which will be cut by the restriction enzyme
used. This latter approach is advantageous as it can link this approach to a
Southern blot RFLP approach as outlined earlier. The information content may
be high or low, depending on the number of inserts of the element targeted
and the frequency of recognition sites of the restriction enzyme used.
Directed PCR: This would involve the use of specific primers, targeted to one
or more known polymorphic loci (see also Sch16tterer and Zangerl, this vo-
lume). These loci may be coding regions [15, 29] or repetitive elements [1, 11,
17]. The information gained per primer pair will clearly be low, but can still be
extremely useful. This technique may be particularly beneficial if a known gen-
otypic variant has been linked to a phenotype. In this case, a pair of primers
may be used, and standard PCR conditions would prevail for that set of pri-
mers. lt is theoretically possible to consider multiplex PCR, where multiple
pairs of primers with no cross-reactivity may be used to generate a DNA profile
of a limited number of loci. The interpretation of the results obtained using this
technology is usually unequivocal, since fragment intensity is even and the
number of variants limited. Furthermore, since the precise locus is known in
each case and there are a limited number of variants, nomenclature may be
quite simple and used for interlaboratory comparisons.
DNA Fingerprinting of Prokaryotic Genomes 9

2.2 Other methods

While RFLP or peR methods are arguably the most commonly used to generate
DNA profiles or fingerprints, other methods have been used and may well be
used more frequently in future. The use of microchip arrays [23] is likely to
prove helpful for genotype analysis. Since this technology is not yet freely avail-
able, it will not be dealt with in detail. However, a simpler version of this will
be described, known as spoligoblotting or reverse line blotting [4, 22, 34, 35].
In this case, detailed knowledge of segments of the genome of the organism to
be studied is required. It has been applied to the genome where a repeat ele-
ment is interspersed with short but variable intervening sequences. Oligonu-
cleotides equivalent to these variant intervening regions can be synthesized
and placed on a linear array on a nylon membrane. Thus, on a membrane
measuring 15 cm by 15 cm, 44 columns of different oligonucleotides may be
spotted using a slot blotter (Miniblotter MN45, Immunetics), with 44 slots of 13
cm in length. (The genome of the organism to be analysed may then be la-
belled. This is most simply done by peR amplification of the entire area con-
taining the interspersed spacers, with biotin [as an example]. Detection may
therefore carried out with peroxidase-labelled streptavidin.).
The apparatus with the slots is rotated 90°, and 44 different labelled gen-
omes are loaded into the slots for hybridization follOwing standard procedures.
Thus, on an apparatus of this size, 44 samples may simultaneously be hybri-
dized to 44 different oligonucleotides. Since the result of this hybridization is
scored only as positive or negative for each spot (or oligonucleotide), the result
is unambiguous, not subject to migration problems and other electrophoretic
or peR variables, and can be scored and compared very easily on a spread-
sheet. The technique has the advantages of being relatively fast and specific,
and the membrane can be reused a number of times.
Spoligoblotting has been used to type M. tuberculosis strains and appears to
offer an intermediate level of discrimination in comparison to a variety of other
probes for this organism. The technique is adaptable to other organisms and
could be used to study multiple loci provided that the specific activity of the la-
belled genome is adequate.

3 Results and Discussion

3.1 Data interpretation and analysis

For all procedures requiring a restriction enzyme digest, it should be noted

that bacterial DNA may be extensively methylated. Therefore, the use of
methylation-sensitive restriction enzymes may cause artefactual variations in
the DNA fingerprint due to methylation-differences ([36, 37]. P. van HeIden, M.