-
MOLECULAR BIOLOGICALAND IMMUNOLOGICALTECHNIQUES ANDAPPLICATIONS
FORFOOD CHEMISTS
Bert PoppingEurofins Scientific Group
Yorkshire, England
Carmen Diaz-AmigoU.S. Food and Drug Administration
Maryland, USA
Katrin HoenickeEurofins WEJ Contaminants GmbH
Hamburg, Germany
InnodataFile Attachment9780470524978.jpg
-
MOLECULAR BIOLOGICALAND IMMUNOLOGICALTECHNIQUES ANDAPPLICATIONS
FORFOOD CHEMISTS
-
MOLECULAR BIOLOGICALAND IMMUNOLOGICALTECHNIQUES ANDAPPLICATIONS
FORFOOD CHEMISTS
Bert PoppingEurofins Scientific Group
Yorkshire, England
Carmen Diaz-AmigoU.S. Food and Drug Administration
Maryland, USA
Katrin HoenickeEurofins WEJ Contaminants GmbH
Hamburg, Germany
-
Copyright 2010 by John Wiley &Sons, Inc. All rights
reserved.
Published by John Wiley & Sons, Inc., Hoboken, New
Jersey
Published simultaneously in Canada
No part of this publication may be reproduced, stored in a
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Limit of Liability/Disclaimer of Warranty: While the publisher
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Library of Congress Cataloging-in-Publicatton Data:
Popping, Bert.
Molecular biological and immunological techniques and
applications for food
chemists / Bert Popping, Carmen Diaz-Amigo, Katrin Hoenicke.
p. cm.
Includes index.
ISBN 978-0-470-06809-0 (cloth)
1. FoodAnalysis. 2. Molecular biology. 3. Immunoassay. I.
Diaz-Amigo,
Carmen. II. Hoenicke, Katrin. III. Title.
TX545.P67 2009
6640. 07dc222009009723
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com
-
To Sue Hefle
Sue Hefle was an internationally recognized food scientist with
major contributions in
the area of Food Allergy and Food Allergens where she was
considered a pioneer. She
was the recipient of numerous national and international awards
and because of her
expertise she was member of numerous advisory panels, task
forces, and working
groups. During her career at the University of Nebraska
sheworked very closely with the
food industry, governments, and consumer organizations.
Unfortunately, we lost her in
2006 at the age of 46 after a long fight against cancer. She
fought the disease with the
same energy and positive attitude she showed during her
professional career. To
recognize her hard work and significant contributions in the
field of Food Science and
in particular Food Allergenssomething she did with passionwe are
dedicating this
book to Sue, our friend and colleague.
-
CONTENTS
CONTRIBUTORS xi
PREFACE xiii
PART Ia MOLECULAR BIOLOGICAL METHODS:
TECHNIQUES EXPLAINED
1. Molecular Biology Laboratory Layout 3Rainer Schubbert
2. Polymerase Chain Reaction 41Hermann Broll
3. Quantitative Real-Time PCR 59Hermann Broll
4. Polymerase Chain ReactionRestriction Fragment Length
Polymorphism Analysis 85Klaus Pietsch and Hans-Ulrich
Waiblinger
5. Single-Stranded Conformation Polymorphism Analysis 105Hartmut
Rehbein
6. Sequencing 119
Rainer Schubbert
PART Ib MOLECULAR BIOLOGICAL METHODS:
APPLICATIONS
7. Meat 135
Ines Laube
8. Genetically Modified Organisms 157Bert Popping
vii
-
9. Detection of Food Allergens 175Carmen Diaz-Amigo and Bert
Popping
10. Offal 199Neil Harris
11. Aquatic Food 209Hartmut Rehbein
PART IIa IMMUNOLOGICAL METHODS: TECHNIQUES
EXPLAINED
12. Antibody-Based Detection Methods: From Theory to Practice
223Carmen Diaz-Amigo
PART IIb IMMUNOLOGICAL METHODS: APPLICATIONS
13. Animal Specification in Speciation 249Bruce W. Ritter and
Laura Allred
14. International Regulatory Environment for Food
Allergen Labeling 267
Samuel Benrejeb Godefroy and Bert Popping
15. Japanese Regulations and Buckwheat Allergen Detection
293Hiroshi Akiyama, Shinobu Sakai, Reiko Adachi, and Reiko
Teshima
16. Egg Allergen Detection 311Masahiro Shoji
17. Soy Allergen Detection 335Marcello Gatti and Cristina
Ferretti
18. Milk Allergen Detection 349Sabine Baumgartner
19. Gluten Detection 359Ulrike Immer and Sigrid
Haas-Lauterbach
20. Nut Allergen Detection 377
Richard Fielder, Warren Higgs, and Katie Barden
21. Fish Allergen Detection 407Christiane Kruse Fste
22. Lupin Allergen Detection 423Christiane Kruse Fste
viii CONTENTS
-
23. Mustard Allergen Detection 445Anne E. Ryan and Michael S.
Ryan
24. Celery Allergen Detection 451Charlotta Engdahl Axelsson
INDEX 459
CONTENTS ix
-
CONTRIBUTORS
ReikoAdachi, Division of Novel Foods and Immunochemistry,
National Institute of
Health Sciences, Tokyo, Japan
Hiroshi Akiyama, Division of Novel Foods and Immunochemistry,
National
Institute of Health Sciences, Tokyo, Japan
Laura Allred, ELISA Technologies, Inc., Gainesville, Florida
Charlotta Engdahl Axelsson, Eurofins Food & Agro Sweden AB,
Ldinkoping,Sweden
Katie Barden, Tepnel Research Products and Services, Deeside
Industrial Park,
Flintshire, UK
Sabine Baumgartner, Center for Analytical Chemistry, University
of Natural
Resources and Applied Life Sciences, Tulln, Austria
Hermann Broll, Bundesinstitut fur Risikobewertung, Berlin,
Germany
CarmenDiaz-Amigo, Center for Food Safety and Applied Nutrition,
U.S. Food and
Drug Administration, Maryland, USA
Christiane Kruse Fste, National Veterinary Institute, Oslo,
Norway
Cristina Ferretti, Microbiotech Department, Neotron S.p.a.,
Modena, Italy
Richard Fielder, Tepnel Research Products and Services, Deeside
Industrial Park,
Flintshire, UK
Marcello Gatti, Microbiotech Department, Neotron S.p.a., Modena,
Italy
Samuel Benrejeb Godefroy, Food Directorate, Health Products and
Food Branch,
Health Canada, Ottawa, Ontario, Canada
Sigrid Haas-Lauterbach, R-Biopharm AG, Darmstadt, Germany
Neil Harris, LGC Limited, Teddington, Middlesex, UK
Warren Higgs, Tepnel Research Products and Services, Deeside
Industrial Park,
Flintshire, UK
Katrin Hoenick, Eurofins WEJ Contaminants GmbH, Hamburg,
Germany
Ulrike Immer, R-Biopharm AG, Darmstadt, Germany
xi
-
Ines Laube, Institut fur Lebensmitteltechnologie und
Lebensmittelchemie,Technische Universitat Berlin, Berlin,
Germany
Klaus Pietsch, Chemisches und Veterinaruntersuchungsamt
Freiburg, Freiburg,Germany
Bert Popping, Eurofins Scientific Group, Pocklington, Yorkshire,
UK
Hartmut Rehbein, Max Rubner-Institut, Hamburg, Germany
Bruce W. Ritter, ELISA Technologies, Inc., Gainesville,
Florida
Anne E. Ryan, ELISA Systems Pty. Ltd., Brisbane, Queensland,
Australia
Michael S. Ryan, ELISA Systems Pty. Ltd., Brisbane, Queensland,
Australia
Shinobu Sakai, Division of Novel Foods and Immunochemistry,
National Institute
of Health Sciences, Tokyo, Japan
Rainer Schubbert, Eurofins Medigenomix GmbH, Ebersberg,
Germany
Masahiro Shoji, Morinaga Institute of Biological Science, Inc.,
Yokohama, Japan
Reiko Teshima, Division of Novel Foods and Immunochemistry,
National Institute
of Health Sciences, Tokyo, Japan
Hans-Ulrich Waiblinger, Chemisches und Veterinaruntersuchungsamt
Freiburg,Freiburg, Germany
xii CONTRIBUTORS
-
PREFACE
From a historical point of view, the analysis of food is
performed predominantly usingtypical chemical or physicochemical
methods. These are, for example, wet chemicalmethods for proximity
analysis or chromatographic methods for the analysis ofpesticides
and veterinary drug residues. In addition, food chemists use
suchphysics-based methods as viscosity measurement and atomic
absorption spectroscopyfor the analysis of heavy metals.
Food chemists in general tend to have good knowledge in some
areas of biologicalanalysis: in particular, regarding methods for
the detection and enumeration ofmicroorganisms and enzymatic
methods for the analysis of single sugars or nitrite/nitrate. In
the past, use of these methods was sufficient for compliance with
regulationsand for quality assurance of food. However, in recent
years several new issues havearisen in thefield of food analysis
which cannot be solved simply by applying chemicalmethods.
Companies and regulators alike now have higher demands for safer
food andproduct quality. This is reflected in more stringent
customer product specifications aswell as new regulations issued
across the world. An example is an allergen-labelingregulation
introduced in the United States on January 1, 2006. Similar
regulations withan expanded scope have come into force in Europe
and, some years ago, in Japan.Another example is the introduction
of regulations for the labeling and traceability ofgenetically
modified organisms. Although at present these are not regulated in
theUnited States, regulations exist in Europe, Japan and other
Asian countries, and inother parts of the world.
Other regulations cover the protection of consumers from
deception by mislabelingor adulteration of products. Examples are
the adulteration of mandarine/tangerinejuice, mislabeling of
premium products such as Angus beef, and the protection ofethnic
minorities from eating products forbidden by their religion. These
analyticalchallenges can be solved easily using methods based on
molecular biological orimmunological principles: polymerase chain
reaction (PCR), real-time PCR, andrestriction fragment length
polymorphism, or in the immunological field, enzyme-linked
immunosorbent assay and dot and Western blot, to name but a
few.
For the typical food chemist this tends to be a generally new
field, since molecularbiological and immunological methods are
based on other principles. But especiallyover the past few years,
borderlines between biological and physicochemical tech-niques have
moved and fields previously separate have amalgamated somewhat
astechniques have been combined tomake it possible to provide
answers faster andmore
xiii
-
cost-efficiently. One example is the invention of biochips,
which, packed withantibodies, are being used to determine amounts
of veterinary drug residues such aschloramphenicol, which until
recently could only be determined by gaschromatographic or
high-performance liquid chromatographical methods. Suchmethods
serve not only to answer questions faster but also complement each
otherby confirming results through a completely
independentmethod.As biologicalmethodsgain more importance in food
analysis, it is prudent for the food chemist to becomefamiliarwith
the techniques and to know the advantages and disadvantages, the
fields ofuseful application, and the pitfalls of biological
methods.
For scientists with a basic chemical education, the contributors
provide, in a simpleand understandable but still
comprehensivemanner, descriptions of themost importantmethods used
in routine molecular biology and immunology and give
selectedexamples of important applications of these techniques in
food analysis. The bookis aimed at students and professional food
chemists as well as quality assurancemanagers and can serve as
guidance in understanding the techniques as well asimplementing
them in a laboratory to expand and complete a service
portfolio.
BERT POPPING
Yorkshire, England
CARMEN DIAZ-AMIGO
Maryland, USA
KATRIN HOENICKE
Hamburg, Germany
Note: Several of the figures that appear in the book may be
viewed in color at
ftp://ftp.wiley.com/public/sci_tech_med/molecular_biological.
xiv PREFACE
-
PART Ia
MOLECULAR BIOLOGICAL METHODS:TECHNIQUES EXPLAINED
-
CHAPTER 1
Molecular Biology Laboratory Layout
RAINER SCHUBBERT
Eurofins Medigenomix GmbH, Ebersberg, Germany
1.1 INTRODUCTION
In this chapter methods for the analysis of biological samples
using molecularbiological methods are described. The main focus
will be on topical methods usedin routine laboratories. However,
the developmental rate of analytical methods andinstruments is high
in this field, and every year new applications are established
inroutine laboratories. Perhaps in a few years some types of
routineDNA analysis will beperformed with transportable instruments
directly in food production facilities or foodstores. The
applications described herein are examples that represent the wide
field ofanalyses performed by molecular biological methods in daily
analysis work. General-ly, the success of the analysis depends on
correct sampling and storage, the DNAcontent of the sample, the
correct DNA extraction method, and the correspondinganalysis
method. All methods described here are based on polymerase chain
reaction(PCR), which is described later. For some of the analyses
described, the methods aredefined by legislation, for some analyses
commercially available kits can be used, andfor other analyses
in-house methods must be developed and validated directly in
thelaboratory.
The protocols for DNA extraction depend on the method used and
are availablefrom the manufacturer of the respective kit. Also, PCR
reaction mixes and cyclingconditions are specific for each assay
and therefore are not described here. Generally,guidelines for
forensic labs describe a very high standard and are therefore
recom-mended (ILAC, 2002).
Molecular Biological and Immunological Techniques and
Applications for Food ChemistsEdited by Bert Popping, Carmen
Diaz-Amigo, and Katrin HoenickeCopyright 2010 John Wiley &
Sons, Inc.
3
-
1.2 LABORATORY
The laboratory design depends on the type of analysis performed.
Detailed recom-mendations for a laboratory design are available,
for example, at
www.ilac.org/publicationslist.html,www.dach-gmbh.de/,
andwww.eurachem.ul.pt/. In this chapter,only principles are
explained. Generally, a molecular biological laboratory should
beseparated into three departments (pre-PCR, thermocycler,
post-PCR), as shown inFigure 1.1.
1.2.1 Pre-PCR Department
At least three different rooms are necessary:
. Room 1: Sample registration. In this room the biological
samples are registered(e.g., barcoded) and subsamples are taken if
necessary.
. Room 2: DNA extraction. In this room the DNA is extracted. All
working stepsshould be performed with filter pipette tips. Coats
and gloves must be worn toprotect both the lab personal and the
samples. Air conditioning is recommended.Forworkwith sampleswith
small amounts ofDNA, a specific portion of the room
FIGURE 1.1 A molecular biological laboratory should be separated
into three departments:pre-PCR (rooms 1 to 3), thermocycler (room
4), and post-PCR (room 5). A computer for sampletracking should be
present in every room. In rooms where work with liquids is
expected, a basinor separate waste bin is recommended. Equipment
such as laminar flow or thermocycler arepositioned in the
schematic. Freezers or refridgerators should be planned depending
on thenumber of samples expected in every room.
4 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
should be separated off. If samples with infectious content are
expected, laminarflow should be present.
. Room 3: PCR setup. In this room the PCR reaction is pipetted
using filter tips. Ifpossible, all PCR reagents are pipetted on one
bench and the genomic DNA isadded on a second bench to avoid
contamination of the PCR reagents with theDNA. Air conditioning is
recommended.
After each working step the benches have to be cleaned with
suitable reagents. It isabsolutely necessary that no PCR products
be treated in one of these rooms. Ifcontamination has occurred, all
surfaces, instruments, and coats must be cleaned, andall chemicals
and working solutions must be exchanged.
1.2.2 Thermocycler Department
PCRcycling is performed in the thermocycler department
(room4).No plasticmaterialor solutions should be transferred from
this department to the pre-PCR department. Airconditioning is
recommended.
1.2.3 Post-PCR Department
In the post-PCR department (room 5) PCR products are handled
using agarose gelelectrophoresis, capillary electrophoresis, and
other procedures. For this work aseparate set of pipettes, plastic
material, gloves, and coats are necessary. Air condi-tioning is
recommended and is required if the analysis is performed using
geneticanalyzers with a laser and CCD (charge-coupled device)
camera as the detectionsystem. As mentioned above, instruments,
coats, single-use plastic, and all othermaterials must not be
transferred from this department into the pre-PCR department.If it
should be necessary to reamplify PCR products, the PCR master mix
has to beprepared in the pre-PCR department, transferred to the
post-PCR department, and thePCR product added here.
1.3 METHODS
1.3.1 Collection of Samples and Storage of Sample Material
One major aspect of the success of the analysis is correct
sampling of the biologicalmaterial and storage of the samples. Any
mistake at this point would deeply influencesucceeding steps of the
analysis and could lead to a complete failure or to
incorrectanalysis results. Even if DNA extraction from clotted
blood, decomposed meat, orswabs overgrown with fungi might be
successful, it should only be used in forensiccasework or in cases
where no other biological material is available. Swabs for
DNAextraction should be air-dried after sampling. Swabs stored in
any gel or liquid (swabsfor cultivation of bacteria) must not be
used. The best storage conditions for biologicalmaterials are
listed in Table 1.1. For all biological material, freezthaw cycles
should
METHODS 5
-
TABLE 1.1 Sampling and Storage Conditions for Biological
Material
Biological Material Sampling and Storage Conditions
Liquid blood for DNAextraction
Preserved with EDTA (first choice) or heparin (secondchoice);
short-term storage and transport at 4C,long-term storage at 20C.
Avoid thawing andfreezing cycles; transport at20C; thawing in a
waterbath at 37C directly before DNA extraction.
Liquid blood for RNAextraction
Preserved with specific buffers [e.g., RNAlater solution(content
of Qiagen RNA extraction kits)], storage at80C; transport on dry
ice.
Blood spots (only for DNAextraction)
Blood up to 200mL dropped on filter paper [e.g., FTA,FTA-elute
(Whatman)]; long-term storage under dryconditions at room
temperature.
Fresh meat, fish meat, meatfrom seafood
Short-term storage (up to 24 h) at 4C, long-termstorage and
transport longer than 24 h at20C. Strictlyavoid thawing of frozen
material.
Swabs from surfaces,buccal swabs
10 to 15min drying at room temperature after sampling;storage
and transport under dry conditions at roomtemperature.
Bones, teeth, connectivetissues
Short-term storage at 4C, long-term storage 20C.Avoid thawing
and refreezing.
Sperm samples (conservedfor artificialinsemination)
Storage and transport frozen in liquid nitrogen. Iftransported
at 4C or 20C, do not store again inliquid nitrogen.
Dried sperm spots Dry and protect from light at room
temperature.
be avoided. If a frozen sample has thawed and has been refrozen,
the laboratorymust beinformed in order to choose the most suitable
DNA extraction method.
Example 1: If a frozen sperm sample normally used for artificial
insemination wasthawed and later refrozen in liquid nitrogen, the
heads of the sperm cellsmay have beendestroyed. In routine
protocols for DNA extraction from sperm cells a special step
isincluded to pretreat the heads. After this step the used buffer
is not reused in laterextraction steps because for a native sperm
sample the DNA is in the pellet and not inthe buffer. In contrast,
for thawed and refrozen samples, most of theDNA can be in
thisbuffer and therefore DNA extraction from the pellet would
fail.
Example 2: Experiments have shown that fish meat frozen directly
after capturecontains sufficient amounts of DNA for analysis in
about 100mg of sample. But afterthawing and refreezing, 1 to 2 g of
fish is needed to obtain sufficient amounts of DNA;analysis with
only 100mg could fail. However, for meat frommammals, the
influenceis not that strong.
During the samplingprocess,wearing ofgloves, cleaningof
instruments, and theuseof single-usematerial is strictly enforced
toavoidcontamination.Especially for samples
6 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
with a low DNA content (e.g., decomposed samples, degraded
samples, bones, teeth),the risk of contamination is high and can
lead to incorrect or unreliable results.
1.3.2 DNA Extraction
In recent years, several methods for the isolation ofDNA from
biological material havebeen developed, and kits are commercially
available. The method used depends on theconsistency of the
biological material, the ratio expected for the amount of DNA
peramount of biological material, the potential presence of PCR
inhibitors in thebiological material, and the instruments or
pipetting machines present in thelaboratory.
Treatment of the sample with proteinase K and an EDTA buffer,
followed byextraction with phenol and chloroform and ethanol
precipitation of the DNA, leads tovery pure DNA but has all the
disadvantages inherent in handling organic substances.Therefore,
most of the kits available work without phenol and chloroform.
Theprinciple of these kits is treatment of the sample with a
low-salt lysis buffer whichcontains proteinase K and the addition
of a binding buffer containing a chaotropic salt.In the presence of
the correct concentration of the salt (e.g., guanidinium
thiocyanate)the DNA binds to silica which is fixed on membranes
(column-based DNA extractionkits) or coated on magnetic beads.
Proteins, salts, and other components from thebiological material
do not bind to silica. After different washing steps, the DNA
iseluted into water or TE buffer. Other kits are based, for
example, on the characteristicsof DNA at various levels of pH
(Charge Switch, Invitrogen). Kits for low throughput,where all
steps are processed manually, to kits for high throughput, where
most or allsteps are processed on pipetting machines, are available
from most suppliers (seeTable 1.2).
1.3.3 Measurement of DNA Concentration
For a successful analysis it is necessary to determine the DNA
concentration. Thepresence of high concentrations of DNA can
influence downstream applications,which can lead to a total or
partial inhibition of PCR, especially for commerciallyavailable
multiplex PCR kits. For DNA concentrations greater than 10 ng/mL,
themeasurement of DNA concentration by a determination of OD
(optical density) 260/280 nmwith a photometer will lead to reliable
results.With thismethod all DNA that ispresent in the solution is
measured. This is sufficient with DNA from fresh blood ormeat
samples, for example. If an analysis were performed to prove the
identity of adegraded tissue sample, it would be necessary to
determine separately the amount ofDNA from the tissue and from
bacteria and fungi grown on this tissue. These methodsare described
in Section 1.3.8.
1.3.4 Variants in the Sequences of Genomic DNA
The DNA of higher organisms is separated into DNA located in the
nucleus (genomicDNA) and DNA located in the mitochondria (mtDNA).
The genomic DNA isseparated on the chromosomes. At every somatic
cell two copies of the autosomal
METHODS 7
-
TABLE1.2
KitsforDNA
Extractiona
Supp
lier/
Biological
Material
Liquid
Blood
Blood
onFilterPaper
Animal
Tissueor
Meat
Bon
esand
Teeth
Plant
Material
Swabs
Hom
epage
MN
NucleoS
pin
Blood
(740
951.10
/.50/
.250
)
NucleoS
pin
Tissue
(740
952.10
/.50/
.250
)
NucleoS
pin
Tissue
(740
952.10
/.50/
.250
)
NucleoS
pinDNA
Trace
(740
942.4/.25)
NucleoS
pin
PlantII
(740
770.10
/.50/
.250
)
NucleoS
pin
Tissue
(740
952.10
/.50/
.250
)
www.m
n-net.com
NucleoS
pin
Blood
L(740
954.20
)
NucleoS
pin8
Trace
(740
722/.1)
NucleoS
pin8
Tissue
(740
740/.5)
NucleoS
pin
Trace
Bon
eBufferSet
(740
943.25
)
NucleoS
pin
PlantL
(740
539.20
)
NucleoS
pin8
Trace
(740
722/.1
)
NucleoS
pin
Blood
XL
(740
950.10
/.50)
NucleoS
pin96
Trace
(740
726.2/.4)
NucleoS
pin96
Tissue
(740
741.2/.4/.2
4)
NucleoS
pin
PlantX
L(740
540.6)
NucleoS
pin96
Trace
(740
726.2/.4)
NucleoS
pin8
Blood
(740
664/.5)
NucleoM
ag96
Trace
(744
600.1/
.4/.2
4)
NucleoM
ag96
Tissue
(744
300.1/.4/.2
4)
NucleoS
pin8
Plant
(740
662/.5)
NucleoM
ag96
Trace
(744
600.1/.4/.2
4)NucleoS
pin96
Blood
(740
665.1/
.4/.2
4)
NucleoS
pinFo
od(740
945.10
/.50/
.250
)
NucleoS
pin96
Plant(74
0661
.2/
.4/.2
4)NucleoM
ag96
Blood
(744
500.1/.4/.2
4)
NucleoS
pin8Fo
od(740
975/.5
)NucleoM
ag96
Plant(74
400.1/
.4/.2
4)NucleoS
pin96
Food
(740
976.2/.
4/.24)
8
-
Qiagen
QIA
ampDNA
Blood
Kits
(Mini,Midi,
Maxi)
QIA
amp96
DNA
Blood
Kits
QIA
ampDNA
Blood
Mini
Kits
QIA
ampDNA
Micro
Kit
Generation
Capture
CardKit
DNeasy
Blood
andTissue
Kit
QIA
ampDNA
Micro
Kit
DNeasy
Plant
MiniK
itQIA
ampDNA
MiniK
itswww.qiagen.
com
EZ1DNA
Blood
Kits
(200
or35
0mL)
DNeasy
Blood
andTissue
Kit
DNeasy
Plant
MaxiK
itQIA
ampDNA
Micro
Kit
QIA
ampDNA
Blood
BioRob
otMDxKit
QIA
amp96
DNA
Swab
BioRob
otKit
GentraPu
regene
Blood
Kits
FlexiGeneDNA
Kits
Generation
Capture
Kits
Prom
ega
DNA
IQSy
stem
WizardGenom
icDNAPu
rificatio
nKit
DNA
IQSy
stem
Ready
Amp
Genom
icDNA
Purificatio
nSy
stem
WizardSV
Genom
icDNA
Purificatio
nSy
stem
DNA
IQSy
stem
WizardGenom
icDNAPu
rificatio
nKit
DNA
IQSy
stem
www.
prom
ega.
com
ABgene/
Therm
oFisher
Scientific
XK02
-04Genisol
Maxi-Prep
Kit
(singlepreps)
XK02
-04Genisol
Maxi-Prep
Kit
(singlepreps)
XK02
-04Genisol
Maxi-Prep
Kit
(singlepreps)
www.
abgene.
com
(continued)
9
-
TABLE1.2
(Continued)
Supp
lier/
Biological
Material
Liquid
Blood
Blood
onFilterPaper
Animal
Tissueor
Meat
Bon
esand
Teeth
Plant
Material
Swabs
Hom
epage
Genial
First-DNA
all
tissue10
/50/
100/50
0
First-DNA
all
tissue10
/50/
100/50
0
First-DNA
all
tissue10
/50/
100/50
0
First-DNA
all
tissue10
/50/
100/50
0
First-DNA
all
tissue10
/50/
100/50
0
First-DNA
all
tissue10
/50/
100/50
0
www.
genial.de
TepnelL
ife
Sciences
Nucleon
Phytop
ure
Invitrog
enChargeSwitch
gDNA
Blood
Kits
(96)
ChargeSwitch
Forensic
DNA
Purificatio
nKit(1)
ChargeSwitch
gDNA
Minio
rMicro
Tissue
Kit(1)
DNAzol
Reagents(1)
ChargeSwitch
gDNA
Plant
Kit(1)
ChargeSwitch
Forensic
DNA
Purificatio
nKit(1)
www.
invitrogen.
com
ChargeSwitch
gDNA
Serum
Kits
(1)
PureLink
Genom
icDNA
Purificatio
nKit(1)
(bon
emarrow)
PureLinkPlant
DNA
Purificatio
nKit
(1)
GeneC
atchergD
NA
Blood
Kits
(1)
DNAzol
Reagent
(1)
PlantD
NAzol
Reagent
(1)
DNAzolB
DReagent
(1)
aFo
r1,
8,and96
samples
asno
ted.
10
-
chromosomes are present (diploid chromosome set). Therefore,
from all geneticinformation located on these chromosomes, two
copies (alleles) are present in everycell. From the gonosomal
chromosomes two copies of one variant or one copy of eachof the two
variants is present, depending on the gender of the individual (X
and Ychromosomes in mammals, W and Z chromosome in birds). In the
germ cells only onecopy of the autosomal chromosomes and one
gonosomal chromosome are present. Thegenomic DNA is separated into
introns and exons as shown in Figure 1.2. An exon isany region of
the DNA within a gene that is transcribed to the final messenger
RNA(mRNA) molecule, which is translated into proteins, for example
(Gilbert, 1978).Therefore, mutations in these regions can have a
strong influence on the organismwhere they occur. Examples are
variants in BRCA genes, which lead to a higher risk ofdeveloping
breast cancer in humans; mutations at the PKD 1 gene, which leads
topolycystic kidney disease in cats; or variations at the PrP gene
in sheep or goat, whichleads to higher or lower risk to develop
scrapie after exposure to the infectious agent.Mutation in the exon
regions can be insertions (new bases are added to the
DNA),deletions (single bases up to longer parts of DNA aremissing),
point mutations [single-nucleotide polymorphisms (SNPs)],
duplications, or translocations.
Therefore, most of the DNA sequences of exonic regions are
highly conserved in ananimal or plant species. Some can be used for
animal species determination. Oneexample is SNPs at the
mitochondrial cytochrome b gene, which can be detected, forexample,
by RFLP (restriction fragment length polymorphism) followed by
agarosegel electrophoresis. Depending on the DNA sequence, specific
restriction enzymes cutthe PCR products. From the number and length
of the fragments it is possible toconclude the DNA sequence at the
restriction sites (see Section 1.3.9).
In contrast to these conserved exonic sequences, at intronic
sequences (sections thatare spliced out after transcription but
before the RNA is used) mutations have in mostcases no influence on
the individual in which they occurred and therefore are passed onto
the next generation. Furthermore, insertions, deletions, SNPs,
duplications, andtranslocations exist in the intronic regions. In
addition, regions with repeated DNAmotifs are present, known as
STRs (short tandem repeats) or VNTRs (variable numbertandem
repeats). From the length of the repeated motif they are separated
into
Intron Exon Intron Exon Intron
DNA
RNA
Protein
FIGURE 1.2 Genomic DNA is separated into introns (white) and
exons (black). Sequencesfrom exons were transcribed to RNA and
translated to proteins.
METHODS 11
-
microsatellites and minisatellites. At microsatellites the
repeat motif contains 1 to 5base pairs (bp) (Figure 1.3),
atminisatellitesmore than 15 bp. Even if the repeatmotif isspecific
for any microsatellite (e.g., main motif AGAAn for the human
markerD18S51), incomplete repeats also exist. Alleles with
incomplete repeats are describedas microvariants. At every
microsatellite, different numbers of repeats can be found(e.g., at
the very polymorphic human STR SE33/ACTBP2, there are about
100different alleles with 4 to 50 complete and incomplete repeats
(Schubbert, 2002).For example, at most markers used in routine
analysis for human identification, thedifference between the
shortest and longest alleles is 20 to 30 bp. It is necessary
todistinguish between the nearest possible fragments, which can be
1 bp at severalmarkers. In principle, PCR products can be separated
by highly concentrated agarosegels, combined agarosepolyacrylamide
(PAA) gels, PAA gels, or capillary electro-phoresis with liquid
polymer, as described in Section 1.3.7.
1.3.5 PCR
Since the first publication of the PCR method (Mullis, 1990),
thousands ofapplications for DNA analysis have been developed. The
principle of PCR is shownin Figure 1.4.
1. Melting step. Double-stranded DNA is denatured
(single-stranded) in a firsttemperature step at 94 to 95C for 15 to
30 s.
2. Annealing step. The reaction mixture is cooled down to 48 to
60C for15 to 30 s. At this lower temperature, primers (short DNA
molecules with15 to 40 bp specific for the DNA fragment, which
should be amplified) bind
Intron Exon Intron Exon Intron
DNA
Short Tandem Repeat
Sample A: ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG CG CG CG
TTAAAGCCGATAG 11 Repeats TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC
GC GC GC AATTTCGGCTATC
Sample B: ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG CG
TTAAAGCCGATAG 9 Repeats TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC
GC AATTTCGGCTATC
Sample C: ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG
TTAAAGCCGATAG 8 Repeats TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC
AATTTCGGCTATC
Sample D: ACGTCAGATAGTTGCAT CG CG CG CG CG TTAAAGCCGATAG 5
Repeats TGCAGTCTATCAACGTA GC GC GC GC GC AATTTCGGCTATC
FIGURE 1.3 Microsatellites and other variable regions are
located in introns. In this scheme,four different sequences of a
microsatellite with the dinucleotide motif CG with 11, 9, 8, and
5repeats are shown.
12 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
to the single-stranded DNA. The optimal temperature at this step
depends on themelting temperature of the primers.
3. Elongation step. At 72C, Taq polymerase starts elongation of
the DNA strandin the 50 ! 30 direction, starting from the primer,
for 30 s up to some minutes,depending on the length of the fragment
amplified.
These three steps (cycles) are repeated 30 to 40 times,
depending on the amount ofDNAmeasured at the beginning of the
reaction. The complete analysis runs automati-cally in combined
heatingcooling instruments known as thermocyclers. These
areavailable from a variety of suppliers for the analysis of one up
to 2 384 samples inparallel. In the past year, several
thermocyclers with very high heating and coolingrates have been
developed to reduce the PCR time (Table 1.3). PCR for
microsatelliteanalysis or other multiplex analysis can be
performedwith labeled primers as shown inFigure 1.5. Depending on
the analysis system, different dyes are used which can bedetected
with ultraviolet (UV) or infrared light. Combinations of dyes used
in routineanalysis are listed in Table 1.4.
If only a few samples should be analyzedwith a higher number
ofmarkers, it may bemore economical to elongate a specific primer
with a universal DNA sequence tail.PCRwill than be performedwith a
mixture of the specific primers and a labeled primerthat binds to
the universal tail in a singleplex reaction (Qin et al., 2006). If
oneanticipates that a specific marker set will be used in routine
analysis in the future, itcould be useful to redesign the primers.
With optimized primer sets it is possible toperformmultiplex PCR
reactionswith 10 to 15markers. In this case one specific primerfrom
every marker should be labeled.
Primer
Taq - Polymerase
1
2
3
4
5
FIGURE 1.4 In PCR double-stranded DNA become denaturated (1) to
single strands (2).Sequence-specific primers bind to
single-stranded DNA (3) and Taq polymerase starts theduplication
ofDNA from these primers (4). The next cycle starts with these
duplicated fragments(5).
METHODS 13
-
1.3.6 Agarose Gel Electrophoresis
DNA fragments can be separated by agarose gel electrophoresis
and stained with dyessuch as ethidium bromide or PicoGreen. These
dyes interact with double-strandedDNAand emitfluorescent light
after stimulationwithUV light. As ethidiumbromide is
TABLE 1.3 Thermocycler Suppliers
ManufacturerNumber of SamplesProcessed in Parallel Homepage
ABI 96/384/2 96/2 384 www.appliedbiosystems.comEppendorf Various
devices/
configurations available:www.eppendorf.com
. 0.5-mL reaction volume:16 or 77 samples
. 0.2-mL reaction volume:25 or 96 (tubes or plate)samples
. 384-well formatStratagene (Robocycler) 96
http://www.stratagene.com/
products/displayproduct.aspx?pid260
FIGURE1.5 Dye-labeledDNA fragments are produced by PCRwith
dye-labeled primers (a).The size of two different alleles (b) with
7 or 5 repeats (4 bp) differs from that of 8 bp. By co-separation
of a size standard (c, black lines) and an allelic ladder (c, gray
lines), the size can bedetermined and the alleles determined
correctly.
14 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
carcinogenic and toxic, nitrile gloves should be worn to protect
the hands whenhandling dyes, stained gels, or contaminated buffers.
Depending on the workflow inthe laboratory, the dye is
alreadymixedwith themelted agarose and is present in the gelduring
electrophoresis, or the gel is stained after electrophoresis. Also,
ready-to-useagarose gels are available with or without dyes from
somemanufacturers. If prestainedgels are used, special attention
has to be paid because the buffers and chambers willbe contaminated
with the dye. In this case, strict rules should be established in
thelaboratory and chambers, pipettes, and instruments contaminated
with the dye must becontrolled. Contaminated objects should always
be handled with gloves. Dependingon the size of the DNA, the
concentration of agarose, and its quality, fragments
withdifferences of at least 4 bp can be separated.
Agarose Gel Electrophoresis of Genomic DNAThe concentration of
DNA can be determined by OD measurement. However, thistechnique
gives no information about possible degradation of the DNA, which
it isnecessary to know for some applications. For these reasons,
agarose gel electrophore-sis of genomic DNA can be performed. A
size marker that covers the sizes expected(up to 40 kb) has to be
co-separated on the same gel. In Figure 1.6, examples ofdifferent
grades of degradation of genomic DNA are demonstrated. If agarose
gelelectrophoresis shows that almost all DNA is degraded to
fragments shorter than400 bp, for example, it will be very
difficult to amplify a PCR fragment of about 450 or1000 bp length,
which is used routinely for the analysis of mtDNA in humans
oranimals.With the information from this agarose gel, the strategy
has to be changed andthe amplification of two or three smaller
fragments would lead to a successful analysis.
Agarose Gel Electrophoresis of PCR ProductsAgarose gel
electrophoresis of PCR products can be performed as quality
controlbefore further analyses. For RFLP analysis or sequencing of
the PCR product, it isnecessary to determine whether a PCR product
is present and howmuch PCR productis present. Therefore, a size
marker that again covers the expected fragment sizes(at PCR
products, normally about 100 to 1200 bp) with known concentration
has to beco-separated (see Figure 1.12). After detection of PCR
products and estimation of theconcentration, the following analyses
will be more successful because optimalamounts of DNA can be
applied to downstream reactions. For proof of the presenceof fungi,
bacteria, or viruses, PCR followed by agarose gel electrophoresis
issometimes sufficient for diagnosis. This is the case if the PCR
product is specific
TABLE 1.4 Dye Sets Used in Routine Analysis on ABI Genetic
Analyzers
Filter Set/Channel Blue Green Yellow
Orange (Used withFive-Dye Sets)
Red (Used as InternalSize Standard)
D FAM HEX NED ROXF FAM JOE NED ROX
FAM JOE TMR RXNG5 FAM VIC JOE PET LIZ
METHODS 15
-
for the organism, all controls show the results expected, and
subtyping is not necessary(e.g., detection of Chlamydia).
1.3.7 PAA Gel Electrophoresis and Capillary Electrophoresis
Today, high-throughput microsatellite analysis is performed with
PAA gels orcapillary electrophoresis (CE) with automated
instruments (Table 1.5). When usingolder instruments, a swab gel
has to be prepared and the samples must be loadedmanually. In the
first step two panes of glass are treated with NaOH, washed,
fixedtogether, and a PAA solution is placed between the panes.
After polymerization, thepanes are mounted on the instrument and a
pre-run is performed to stabilize theelectrophoresis conditions.
Finally, the samples, mixedwith running buffer, are loadedmanually
to the instrument using an eight-channel pipette. After every run
the glasspanes have to be cleaned and the buffer has to be
exchanged. The advantage of this typeof instrument is better
resolution for specific types of samples and robustness if only
afew runs are performed per week.
With the current generation of capillary electrophoresis
instruments, PCR productsare mixed with formamide and put into the
instrument. Filling the capillaries withviscous polymer, loading
the samples and the size standard to the capillary, and
starting
FIGURE 1.6 Agarose gel picture. The quantity and quality of
isolated genomic DNA can bedetermined in comparison with defined
size standards (lanes 1 and 8) From dried fish (lane 2),degraded
muscle (lane 5), and heart tissue (lane 7) only weak amounts of
mostly degraded DNAcan be isolated. From freshly frozen fish (lane
3) or prawns (lane 4) and bone marrow (lane 6),high-molecular DNA
can be isolated.
16 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
and performing electrophoresis are carried out by the instrument
automatically. In onecapillary analyzer, the ABI 3130 (Figures 1.7
and 1.8), 30 runs of 16 samples with aread length of 600 bp can be
run within 24 h. Routine work for this instrument isreduced to
refilling the buffers and viscous polymers or capillary arrays and
routinecleanup of the instrument (buffer chambers, injection
pumps), which should be carriedout once a week. One disadvantage of
this type of instrument is the aging of thepolymer and array
ifmounted on the instrument, which can be critical if only a few
runsare performed per week.
For PAA electrophoresis or CEwith automated fragment detection
and size calling,the PCR products must be labeled with dyes (Table
1.4). During the establishment of anew assay, for best results
several dilutions of PCR products should be tested afterPCR. For
electrophoresis the PCR products have to be mixed with a loading
dyeaccording to the concentrations given by themanufacturer and an
internal size standardthat is co-separated in every line or
capillary. During electrophoresis a laser stimulates
TABLE 1.5 Instruments for PAA or Capillary Electrophoresis
Manufacturer Instrument
Numbers ofSamplesProcessed
inParallel
Numberof Dyes
Swab Gelor CE Homepage
ABI 3130 XL 96 5 CE www.appliedbiosystems.comAmersham Mega-
BACE96 4 CE www.4.amershambiosciences.
comLiCor 4300 48 2 Swab gel www.licor.com
FIGURE 1.7 Genetic analyzer ABI 3130 with control computer.
METHODS 17
-
the dyes to emit fluorescent light, which is measured by a CCD
camera. This camerascans the detection window 5000 to 10,000 times
per run. Thereby, the data collectionsoftware of the instrument
collects the data of four or five different dye channels.
Bycomparing the raw data from the red channel (on ABI instruments,
the internal sizestandard is recorded in the red channel) with data
from the other channel, the analysissoftware (e.g., GeneScan or
Genemapper for instruments from Applied Biosystems)calculates the
fragment size of the PCR products (Figure 1.9).
For a reproducible allele calling, categories can be defined by
the software (forinstruments from Applied Biosystems, e.g.,
Genotyper or GeneMapper) for everymarker at additional analysis
steps which can be used for further analyses. Dependingon the
instrument and size standard used, the same allele/PCR fragment can
be definedwith different lengths (Figure 1.10). Therefore, it is
necessary to standardize the resultsfor intra- and interlaboratory
data exchange.
For some commercially available microsatellite multiplex PCR
kits, allelic laddersare available (Figure 1.11). These ladders
should be analyzed within every run in aseparate line or capillary
to assure the quality of the analysis. For individualmarker setsit
is recommended that an allelic ladder be developed or at least that
one or two controlsamples with a known genotype be analyzed in
every batch of samples. For somemarker sets, ring trialswere
organized [e.g., by ISAG (International Society for AnimalGenetics)
for horse, cattle, sheep, goat, dogs, and cats; and by ISFG
(InternationalSociety for Forensic Genetic) and DGRM (German
Society for Legal Medicine) forhumans].
FIGURE 1.8 Detailed view of ABI 3130. Liquid polymer stored in a
bottle (a) becametransported to a capillary array (b) by a pump
(c). Samples prepared for electrophoresis becamestored in a tray
(d). By electrophoresis, PCR products migrate to the detection
window (e) andbecome measured by a CCD camera.
18 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
FIGURE 1.9 Data from GeneScan analysis. Size of PCR products
(blue, green, and blackpeaks) is measured by comparison with an
internal size standard co-separated in the samecapillary. (a) Size
standard ROX500 fromABI; (b) PCR fragments and size standard. (See
insertfor color representation.)
FIGURE 1.10 Comparison of data from Genotyper analysis.
Identical PCRwas co-separatedwith size standard ILS600, Promega (a,
b) andwith size standardROX500 (ABI) (c, d) in parallelon an ABI
3100. Using ROX500, fragment lengths seems to be 2 to 3 bp longer
than when usingILS.
METHODS 19
-
1.3.8 Real-Time PCR
For some applications it is necessary to determine the amount of
specific DNA or RNAin a biological sample. At the beginning of
every PCR reaction only a few amplifiedfragments are present. In
the following logarithmic phase the number of PCR productsis
doubled in every cycle under optimal conditions. Depending on the
amount of DNAat the beginning of the reaction, after various cycles
the reaction reaches the plateauphase. This is influenced by
several factors. During PCR the amount of free
nucleotidesavailable, which are necessary to synthesize a newDNA
strand, and the amount of freeprimers decrease. At a later time,
high numbers of PCR fragments are present. Thesefragments also
reanneal and are not available to bind primers. Finally, the
efficiency ofthe enzyme weakens from cycle to cycle.
Applying PCR with, for example, 35 cycles and a separation on
agarose gel, nodifference can be detected, whether 10, 25, 50, or
100 ng of genomic DNA is analyzedin the PCR reaction (Figure 1.12).
In contrast, using real-time PCR it is possible todistinguish
between the various amounts of DNA (Figure 1.13). Real-time PCR can
becarried out with a pair of unlabeled primers and the presence of
SybrGreen, whichemits fluorescent light after stimulation when
double-stranded DNA is present.However, SybrGreen also binds to the
high-molecular DNA that is added to the PCRreaction, to
primerdimmers, and to unspecific PCR products. As a consequence,
onlyone PCR product can be detected per reaction. Therefore, during
the developmentphase of a new assay, the PCR products should be
controlled by an agarose gelelectrophoresis after real-time PCR.
The optimal amount of high-molecular DNAadded to the PCR also has
to be determined. The performance of a melting curve afterthe PCR
reaction can assure that the PCR products expected are
amplified.
FIGURE 1.11 Comparison of data from Genotyper analysis.
Comparing PCR products withfragments of allelic ladders, intra- and
interlaboratory data exchange is possible. Upper panel:allelic
ladder of markers D3S1358, TH01, and D18S51 used for human
identification shown; atthe lower panel: DNA profiles from four
different DNA samples.
20 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
FIGURE 1.12 Agarose gel picture. In comparism with defined size
standard (lane 1) thequantity and fragment size length of PCR
products can be determined. Different amounts ofDNA (60 ng, 30 ng,
15 ng, 7.5 ng, 3.75 ng, 1.8 ng, and 900 pg; lanes 2 to 8) were
analyzed byreal-time PCR with primers specific for mitochondrial
DNA. In contrast to online measurementduring real-time PCR by
agarose gel electrophoresis, quantification of the amount of
genomicDNA put into this PCR is not possible.
FIGURE 1.13 Different amounts of DNA (60 ng, 30 ng, 15 ng, 7.5
ng, 3.75 ng, 1.8 ng, and900 pg) were analyzed by real-time PCR with
primers specific for mitochondrial DNA.Depending on the amount of
DNA put into PCR, the amplification curves cross the
threshold(horizontal line) with one cycle difference (upright gray
lines).
METHODS 21
-
Real-time PCR is more specific when primer and labeled probes
are used. By acombination of specific primers and probes with a 10C
higher melting temperature, itis possible to detect, for example,
1-bp mutations (SNPs) by real-time PCR. Differenttypes of probes
are developed. At dual-labeled probes at the 50 end a reporter dye
islabeled, which emits fluorescent light after stimulation. At the
30 end a quencher dye islabeled. If reporter and quencher are
localized nearby, the fluorescent light of thereporter dye is
quenched. The Taq polymerase used for PCR also has
exonucleaseactivity. Therefore, during the elongation phase of the
PCR the probe is not meltedfrom the DNA strand and is destroyed by
the Taq polymerase. The reporter andquencher dyes are separated and
the light emitted from the reporter can bemeasured bythe detection
system of the thermocycler. At the beginning of the reaction
theinstrument measures the background signal from the reporter dye.
During every cyclethe instrument measures the signal intensity and
determines the cycle at which thesignal is significant higher than
the background signal of the samples at the start ofthe analysis.
This cycle is called a cycle of threshold (Ct). HighCt valuesmean
that lessDNA was present at the beginning of the PCR.
Real-time PCR can also be used for the quantitative analysis of
DNA or RNA. Inexpression analysis, the Ct values of a
constant-expressed gene (a housekeeper gene)and a
variable-expressed gene (a gene of interest) are compared.
Expression analysis isnot often used in the analysis ofmeat or food
products. Currently, different instrumentsare available for
real-time PCR. Depending on the manufacturer, the stimulating
lightis emitted from a laser or from a tungsten bulb in combination
with a filter. Thefluorescent light emitted from the reporter dye
is measured through a prism or filtersystem. The number of parallel
dyes detected varies from three to five. Instrumentscurrently
available are listed in Table 1.6.
1.3.9 RFLP Analysis
For RFLP (restriction fragment length polymorphism) analysis,
the qualities ofrestriction endonuleases are used. These cut
genomic DNA or PCR products at ornear specific sequences. At a
single basepair mutation, two different sequences arepresent. The
corresponding enzyme cleaves only one of the two possible DNA
strands.The fragments can be detected by agarose gel analysis.
1.4 APPLICATIONS
1.4.1 PCR and Detection of PCR Fragments
Gender Determination of Animals
Cattle The gender determination of cattle is necessary to answer
two questions:
1. During embryogenesis of twins, anastomotic blood vessels can
be developedbetween the placentas, which can lead to problems with
dioecious twins. Throughthese blood vessels, stem cells and
hormones can be exchanged. If the female twin
22 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
TABLE1.6
Instruments
forReal-Tim
ePCR
Manufacturer
Num
berof
Samples
Processedin
Parallel
Num
ber
ofDyes
Stim
ulating
Light/Detectio
nSy
stem
Performance
ofMeltin
gCurve
Possible?
Hom
epage
ABI
48/96/38
45
LED/Halog
en/
Laser
yes
www.app
liedb
iosystem
s.com
Epp
endo
rf96
Twoop
tions
available:
upto
twoor
four
different
dyes
detectable
96LEDsfor
excitatio
n;channel
photom
ultip
lier
fordetection
yes
www.epp
endo
rf.com
Roche
96/384
4yes
www.ro
che-applied-science.com
Stratagene
(Mx3
005P
)96
5Halogen
lamp;
photom
ultip
lier
yes
www.stratagene.com/qpcr
23
-
receives the anti-Mueller hormone from the male twin, sexual
organ development willbe inhibited. This can range from missing
organs in the newborn calf to functionalproblems even when the
organs are present. In almost all cases such female twins willnot
become pregnant. Development of the sexual organs of the male twin
is notinfluenced. A farmer thus has two choices after the birth of
dioecious twins: He or shecan feed the female calf and slaughter it
as he or shewould amale calf, or can determineby PCR whether blood
cells that carry a Y chromosome are present in the circulatingblood
of the female calf. If these cells are present, it is very likely
that the anti-Muellerhormone was transferred to the female twin.
The analysis has to be performed withEDTA or heparin blood. Hairs
with roots would lead to an incorrect result because thecells
transferred are present only in the blood. The cells transferred
are underrepre-sented when using muscle biopsies and buccal swabs
and therefore may not bedetected.
2. If female calves or cattle are slaughtered, female meat might
be declared to bemale meat, perhaps unintentionally. Because male
meat receives higher prizes andhigher prices are paid for exports,
female meat might intentionally and deceitfully bedeclared to be
male meat.
According to European Commission (EC) Regulation 2002/765/EC of
3/5/2002,the analysis of gender determination has to be performed
by PCRwith primers specificfor DNA fragments which are located on
both the X and Y chromosomes:
. Forward and reverse amelogenin (Ennis and Gallagher, 1994);
the length of aPCR fragment specific for the X chromosome is 280
bp, one specific for the Ychromosome is 218 bp.
. ZFX and ZFY forward and ZFX/ZFY reverse (Zinovieva et al.,
1995); the lengthof a PCR fragment specific for theX chromosome is
132 bp, one specific for theYchromosome is 282 bp.
The use of two primer pairs reduces the risk of an incorrect
result. Sincemutations ofDNA sequences are spread over the complete
genome, a mutation can also be presentat any primer binding site.
If this occurs at the binding site of genes located on theY
chromosome but not on the X chromosome, only the fragment specific
for the Xchromosome is amplified. In this case, male meat can be
determined incorrectly tobe female meat. The use of two independent
DNA fragments for the analysisreduces the risk of a wrong result
dramatically. PCR products can be detected byagarose gel
electrophoresis or capillary electrophoresis (with one labeled
primer perpair). In Figure 1.14, genotypes of a male and a female
sample detected by CE areshown.
Birds Gender determination can be necessary for bird species
without externalgender differences. As surgery (laparatomy) with
anesthesia for gender determinationcan lead to the death of the
birds, DNA analysis from blood or feathers is a
noninvasivealternative. Normally, gender determination by DNA
analysis is not performed for
24 MOLECULAR BIOLOGY LABORATORY LAYOUT
-
poultry such as geese but, rather, for parrots, parakeets, and
some birds of prey. Forgender determination of a wide variety of
birds, except the ratites (ostrich, rhea), whichare sometimes grown
for meat production, universal primers were used (Griffithset al.,
1998). For gender determination of cattle, PCR products can be
detected byagarose gel electrophoresis or CE. In Figure 1.15
genotypes of a male and a femalesample detected by CE are
shown.
Analysis of Special Ingredients in Food ProductsIn modern food
production different groups of additives are used, which
sometimescannot be detected or distinguished by methods other than
PCR. Examples for those
FIGURE 1.14 Gender determination of beef meat. Samples of male
(a) or female (b) originwere analyzed by PCRwith primers specific
for bovine amelogenin locus.With samples of maleorigin, two PCR
products can be detected; with samples of female origin, one PCR
product canbe detected.
FIGURE 1.15 Gender determination of birds. Samples of male
parrot (a) or female parrot (b)origin were analyzed by PCR. With
samples of male origin, one PCR product can be detected;with
samples of female origin, two PCR products can be detected.
APPLICATIONS 25
-
additives are hydrocolloids [e.g., xanthan (E415), guar gum
(E412), or locust beangum (E410)], which are added to products such
as yogurt, ketchup, or instant soup fortechnical reasons. However,
in some cases these additives may not be declared.Xanthan, which is
allowed to be added to organic food, is produced by the
fermentationof wood by Xanthomonas campestris strains. Xanthan can
therefore be detected withprimers specific for the DNA of these
bacterial strains. Cellulose and pectin areproduced from the shells
of apples and citrus fruits. Theoretically, these substances canbe
detected with primers specific, for example, for apple chloroplast
genes. However,experiments have shown that for most samples the DNA
was too degraded during theproduction process for a successful
analysis.
The detection of guar gum (E412) or locust bean gum (E410) is
possible withprimers specific for the DNA of Cyamopsis (guar bean)
and Ceratonia (carob), asdescribed elsewhere (Urdiain et al.,
2004). For the analysis of food products the DNAextraction should
be performedwith specially developed kits.With these types of kits
itis possible, for example, to extract DNA from chocolate, cheese
and other milkproducts, or marmalade without coextraction of such
PCR inhibitors as salts, fattyacids, or humid acids. Examples of
kits are the food kits from MN and Genial(see Table 1.2). For some
highly processed food products the DNA content might bevery low.
For these applications the Funnel Food Kit from MN allows lysis of
thesample material in volumes up to 10mL and the elution of the DNA
into volumes lessthan 100 mL. PCR products can be detected with
agarose gels.
Bacterial Species and Antibiotic Resistance DeterminationAs
described in Chapter 6, determination of bacterial species may be
necessary forpathogen detection in meat and food products:
identification of infectious pathways ofpersonsworking in food
production or other sensitive departments, or for identificationof
bacterial strains used in food production. With sequencing of PCR
products of thebacterial 16S RNA gene, identification is possible
if only one strain is present. Formixed samples, interpretation of
sequencing data can be difficult or impossible. Forthis analysis at
least 6 h is necessary after DNA extraction. In contrast, PCR
withprimers specific for single strains followed by agarose gel
electrophoresis gives resultsin 3 to 4 h following DNA extraction.
Using real-time PCR, results can be available asquickly as 2 h
after DNA extraction. For both applications, kits are available
fromdifferent manufacturers. For several bacterial strains,
sequences of plasmids causingantibiotic resistances are known.
Therefore, PCR detection of specific sequences canreduce the
analysis time and accelerate the therapy.
Example: Testing of Enterococcus against vancomycin and
teicoplanin resistancewith other methods (e.g., VITEK, bioMerieux)
can lead to unclear or variable results.By PCR four different
plasmids that encode for high-level resistance againstvancomycin
and teicoplanin (plasmid VanA), high-level resistance
againstvancomycin and variable resistance against teicoplanin
(plasmid VanB), or low-level resistance against vancomycin, and
practically no resistance against teicoplanin(plasmids VanC 1, VanC
2/3) can be detected specifically (Dutka-Malen et al., 1995;Ballard
et al., 2005).
26 MOLECULAR BIOLOGY LABORATORY LAYOUT
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1.4.2 Microsatellites and Variable Number of Tandem Repeats
DNA ProfilingBy microsatellite analysis DNA fingerprints can be
made from almost all mammals.Primers are published for a wide
variety of mammals, birds, and fishes. For someanimal species used
in agriculture, commercial PCR kits are available (Table 1.7);
forother species, sets of markers are available some of which were
tested in ring trials(sheep, goat, pig). In contrast to the
analysis of human DNA, currently no allelicladders are commercially
available for these marker sets except for porcine microsat-ellite
analysis with a kit from Biotype.
For proof of the identity of animals grown for food production
or of the origin ofmeat, DNA profiling can be used based on several
concepts:
1. DNA profiles from all animals used for breeding are collected
within a database.From every offspring a DNA profile is generated,
compared with the profile of theparents, and put into the database.
For every marker analyzed, the offspring must haveone common allele
with the biological father and one with the biological
mother(Figure 1.16). When, for example, the animal is sold or
transported and there is anydoubt about the identity, a new profile
can be generated from the blood, tissue, or hairs.After the animal
has been slaughtered, a sample is taken from the meat and the
DNAprofile is compared with the existing profile.
2. Fromall sires used for breeding theDNAprofile is collected in
a database.With aspecific type of eartag, a small piece of tissue
is collected during the collecting process.This tissue sample is
stored until the animal loses the eartag. Before the animal gets
anew eartag, the DNA is extracted from the tissue collected earlier
and compared withthe current DNA profile and/or with the profile of
the putative father. After the animalhas been slaughtered, the DNA
profile from the meat can be compared with the profileof the tissue
sample collected earlier.
3. From all newborn animals, tissue is collected as described
above. DNA profilingis performed only if there is any doubt about
the identity of the animal. The sires usedfor breeding are not
tested in this concept.
With all these concepts, controls can be exercised randomly.
Depending on thestatistical concept, the costs for the analysis can
be reduced, but there remains a
TABLE 1.7 Commercially Available Kits for Microsatellite
Analysis
ManufacturerAnimalSpecies
Number ofMarkers
Allelic LadderAvailable? Homepage
AppliedBiosystems
Cattle 11 no www.appliedbiosystems.com/Horse 16 no
Dog 10 no
Finnzymes Cattle no www.finnzymes.fi
Biotype Pork yes www.biotype.de
APPLICATIONS 27
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probability of up to 99% that awrong declaration can be
detected. DNA for the analysiscan be extracted from EDTA blood,
heparine blood, blood spots on filter paper, tissue/meat, bones, or
teeth. It is also possible to extract DNA from hairs with roots,
but hairscan be contaminated with DNA from the saliva of other
animals or with feces. Withmost farm animals, DNA extracted from
buccal swabs contains less genomic DNAfrom the animal. In ruminants
a swab contains a large amount of saliva and bacteriafrom the rumen
but a small amount of cells from the mucosa. In addition,
PCRinhibitors from food may be present. The quantity and the
quality of DNA extractedfrom meat should be determined by OD
measurement and/or agarose gel control.Applying PCRwithmultiplex
kits, the amount of DNA suggested by themanufacturershould be added
to the PCR to provide balanced DNA profiles (Figure 1.17).
Population Genetic or Animal Breed DeterminationFor some reason
it may be necessary to determine whether an individual animal is
partof a specific population or breed. The first step in answering
this question is to definethe population or breed and identify all
animals that are typical for the respective group.DNA profiles can
then be generated and compared.
Example 1: Information was requested from a breeding
organization, the AberdeenAngus Society, as to which sires were
used most often for artificial insemination andnatural
insemination. Sperm samples were collected from these sires and
also from
FIGURE 1.16 Paternity testing. DNA profiles from offspring (a),
dam (b), and two possiblefathers (c, d)with twomarkers specific for
canineDNA (PEZ10 andFH2361) are shown.At PEZ10, offspring and dam
share allele 283. Therefore, allele 299 present in the offspring
has to bepresent in the biological father (present only at sample
c). At FH2361, offspring and dam shareallele 338. Therefore, allele
342 present in the offspring has to be present in the biological
father(present at both samples c and d). Frommarker PEZ10, sample d
could not be from the biologicalfather of sample a.
28 MOLECULAR BIOLOGY LABORATORY LAYOUT
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other breeds present in the same region that could delivermeat
which couldwrongly bedeclared to beAberdeenAngus (AA)meat.
TheDNAwas extracted and analyzedwithabout 120 markers. The allele
frequencies of these 120 markers from the AA samplesand non-AA
samples were compared.Markers were identified that show typical
allelesof the AA samples and other alleles of the non-AA samples.
With these markers, blindtests were performed with meat samples of
known origin. All of these samples weretyped correctly.
Example 2: At a control on a farm, several animals without
eartags were found.Normally, all such animals have to be
slaughtered. According to documents of thefarmer, the biological
mothers of some of the animals were still on the farm, someweresold
or slaughtered, but closely related animals were still present.
Blood samples werecollected from all animals on the farm and DNA
profiles were generated. Parallelsamples from other animals of the
same breed were collected and analyzed. Bycomparison of the DNA
profiles of the offspring with those of the putative parents,some
of the animals could be identified. Profiles from the other animals
were comparedwith profiles of the closely related animals of the
putative mothers and the unrelatedanimals. The likelihood that
these animals could be offspring of the animals asdocumented by the
farmer was calculated.
Example 3: A fish retailer declared smoked salmon to be wild
from salmoncaptured in a specific river. The food analyst has
doubts about this declaration;he thinks that the meat is from
farmed salmon. In this case reference samples must becollected from
salmon captured in the river and from salmon raised on all farms.
Aftermicrosatellite analysis, allele frequencies have to be
determined and the likelihoodhas to be calculated whether the
salmon can be from the river population or from oneof the farm
populations.
Determination of Basmati RiceBasmati rice is a long-grain rice
that grows in the Himalaya region of India andPakistan. Actually,
17 Basmati varieties are recognized (Table 1.8). Since Basmati
rice
FIGURE 1.17 DNA profile with 11 markers specific for bovine DNA.
From left to right: (a)TGLA 227, BM2113, TGLA53, ETH10, SPS115; (b)
TGLA126, TGLA122, INRA23; (c)ETH3, ETH225, BM1824.
APPLICATIONS 29
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is more expensive than other long-grain rice and upon import
into the European Union(EU) lower tax rates have to be paid,
controls are necessary as to whether a samplecontains only Basmati
rice or a mixture with other rice varieties.
ADNAmicrosatellite methodwas developed by the Food
StandardsAgency (FSA)in London to check retail sales of Basmati
rice in the UK market (FSA, 2004). In theUK, Basmati is defined by
a code of practice (COP) agreed to among the rice
industry,retailers, and the enforcement authorities. The COP lists
the varieties that can bedescribed as Basmati (Table 1.8) and
outlines a specification for the rice in which arealistic level of
unavoidable contamination with non-Basmati rice varieties is
set.Contamination can happen during harvesting of the rice,
transport to local traders, andexport into the EU. The
contamination allowed following COP is a maximum of 7%.Because the
detailed analysis protocol may change in the near future and
because it canbe retrieved from the FSA homepage on the web, only
the principle of analysis isexplained.
About 100 g of rice is milled with a coffee grinder. From this
powder DNAshould be extracted in triplicate. From each of the three
DNA samples, PCR isperformed with at least 10 microsatellite
markers (actually, the list of markers RM1,RM16, RM44, RM55, RM171,
RM201, RM202, RM223, RM229, RM241).The genotypes detected are
compared with known genotypes of the approvedvarieties. In
mixtures, contamination with nonapproved varieties is documented.In
2006 a ring trial of the quantitative determination of non-Basmati
rice varietiesin a mixture with Basmati rice varieties was
organized by the FSA. The results from 9of the 11 participating
laboratories differed by less than 0.6% from the weightedmixtures;
two laboratories had bigger differences. This test demonstrated
that labora-tories with experience in microsatellite analysis can
deliver reliable results in analysesof Basmati rice or mixtures of
Basmati- and non-Basmati varieties. In Figure 1.18profiles from
Basmati rice and a mixture of Basmati and non-Basmati varieties
areshown.
TABLE 1.8 Approved Basmati Rice Varieties
Variety from India Variety from India or Pakistan Variety from
Pakistan
Basmati 217 Basmati 370 Basmati 370Ranbir SuperBasmati 370
KernelBasmati 386 Basmati 198Taraori Basmati
385DehradunPusaKasturiMahi SugandaHaryanaPunjab
30 MOLECULAR BIOLOGY LABORATORY LAYOUT
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Similar analyses can also be made on other rice varieties, such
as Jasmine rice.Authentic samples of typical Jasmine rice and of
other rice varieties that can be used tominic Jasmine rice have to
be collected and analyzed with a broad range of markers.The next
step is performed similar to that described earlier for the
establishment of abreed-specific analysis for Aberdeen Angus
cattle.
Basmati and Jasmine rice are both famous for their specific
flavor, caused by amutation at the putative betaine aldehyde
dehydrogenase 2 (BAD2) gene, which canalso be determined by DNA
analysis (Bradbury et al., 2005a, b). As the predispositionflavor
is recessive, only rice grains that are homozygous for themutation
develop theflavor (Figure 1.19)
Identification of Bacterial Strains by VNTR AnalysisFor some
bacterial strains, sequencing of 16S rRNA or real-time PCR with
specificprimers cannot provide all the information needed.
Especially if infectious pathwayshave to be followed, subtyping
with VNTR is the method of choice. For severalbacterial species,
VNTR (and STR) analysis methods are described that can be used.
InFigure 1.20DNAprofiles received byVNTRanalysis ofFrancisella
strains are shown.Themethod can also be used to determine whether
reference strains are pure or containa mixture of two or more
substrains (Bystrom et al., 2005).
FIGURE 1.18 DNA profiles with two markers specific for rice DNA
(left marker, RM171;right marker, RM55: (a, b) two different
mixtures of rice varieties Pusa and Dehradun; (c) puresample of
rice variety Pusa; (d) pure sample of rice variety Dehradun.
APPLICATIONS 31
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1.4.3 Real-Time PCR
Determination of DNA ConcentrationsAs described above, for
several applications it is necessary to determine the
DNAconcentration before PCR. For DNA concentrations higher than 10
ng/mL, measure-ment of the DNA concentration by a determination of
OD 260/280 with a photometerwill lead to reliable results. These
amounts of DNA can be expected after DNA
FIGURE1.19 DNAprofiles with PCR product coding for fragrance.
Rice sample contains (a)fragrant and nonfragrant grains; (b) only
fragrant grains; (c) only nonfragrant grains.
FIGURE 1.20 DNA profiles with two markers specific for
Francisella DNA (left marker,FtM08; right marker, FtM21). Strain
(a) can be distiguished from strains (b) and (d) by markerFtM08 and
from strain (c) by marker FtM21. Strain (b) can be distiguished
from strains (a), (c),and (d) by marker FtM08 and from strains (c)
and (d) by marker FtM21. Strain (c) can bedistiguished from strains
(b) and (d) by marker FtM08 and from strains (a), (b), and (d)
bymarker FtM21. Strain (d) can be distiguished from strains (a),
(b), and (d) by markers FtM08and FtM21.
32 MOLECULAR BIOLOGY LABORATORY LAYOUT
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extraction from blood, fresh tissue samples, and buccal swabs
with high numbers ofattached cells. In Table 1.9 ranges of DNA
concentrations are listed. By ODmeasurement, all DNA present in a
sample is detected. This should be no problemfor fresh-drawn or
optimally stored blood or tissue samples because in this case it
isexpected that only DNA from the donor of the sample is
present.
For older or nonoptimally collected or stored buccal swabs or
decomposed samples,it can happen that a part of the measured DNA
comes from bacteria or fungi. Inaddition, for those samples the DNA
yield expected will be lower than that listed inTable 1.9. Other
biological material, such as teeth, bones, or connective tissue,
orboiled, grilled, or smoked meat, contains less DNA. In this case
a determination ofDNA concentration by real-time PCR is necessary.
Using a real-time instrument it ispossible to determine the total
amount of DNA in a sample or the amount of DNA froma specific
species.
Total DNA Kits are available from some suppliers to determine
the concentration ofDNA or RNA by staining with PicoGreen (e.g.,
Quant-iT PicoGreen dsDNA AssayKit, Invitrogen). The kit also
contains a control DNA of known concentration.Normally, analysis
with this kit should be performed with a fluorometer, but it isalso
possible to perform the analysis on some real-time instruments. For
this, the DNAsolution is mixed with a very low concentration of
PicoGreen and a melting curve isanalyzed (Figure 1.21). By
comparing the signal intensities of controls and samples at
aspecific temperature, the DNA concentration can be determined.
DNA fromVertebrates For the differentiation of DNA from bacteria
and the DNAfrom vertebrates, analysis can utilize primers specific
for genomic or mitochondrialDNA. Real-time PCR with primers
specific for conserved sequences of themitochondrial cytochrome b
gene detects DNA from all mammals and most fishes.For the detection
of chondrichtyes (ray, shark) and prawns, other primers have to
bechosen. The analysis can be performed with unlabeled primers.
Amplicons can bedetected by SybrGreen. For most instruments,
control samples with knownconcentrations can be used as markers,
and using these the DNA concentration ofsamples is calculated
automatically.
DNA from Specific Species For the analysis of genomic human DNA,
kits fromtwo suppliers (i.e., Applied Biosystems, Promega) are
currently available. With these
TABLE 1.9 DNA Yield of Biological Material
Biological MaterialAverage DNA Concentration of Optimally
Stored or Fresh-Drawn Samples
EDTA or heparin blood 5 ng/mL bloodMuscle tissue, liver tissue
4070mg/100mg tissuePlant material 130mg/100mg tissueRice grains
15300 ng/ g grainsProcessed food products 0.15mg/100mg product
APPLICATIONS 33
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kits the detection of concentration of total human DNA or male
human DNA ispossible. Dilutions of the control DNA contained in the
kit allow calculation of theconcentration down to 6 pg/mL, which
corresponds to one genome equivalent. Resultsof analysis with a
Quantifiler kit from Applied Biosystems are shown in Figure
1.22.
Several publications describe assays for genomic animal DNA.
Analysis of shortinterspersed nuclear elements (SINEs) allows
specific detection of DNA from manyspecies, including cattle,
horse, pig, deer, and dog. SINEs are repeated, unblocked,
anddispersed throughout the genome sequences. They represent
retroposons (included inthe genome transcripts of intracellular
RNA) and constitute more than 20% of thegenome of humans and other
mammals. Unique sequences could be identified forevery species and
used for the development of a species-specific PCR assay (Walkeret
al., 2003). For this application the laboratory has to prepare its
own control sampleswith the DNA extracted from reference
samples.
Specific detection of DNA can also be carried out by real-time
PCR with primersspecific for microsatellites. Examples of
microsatellites that are specific to one speciesare listed in Table
1.10.
In principle, all DNA sequences that are known as species
specific can be used forthe development of an assay for the
detection of DNA from specific animals, plants, orbacteria. In
control experiments it must be shown that no other DNA fragments
areamplified using the primers chosen. Generally, assays with
primers and a specific
FIGURE 1.21 Different amounts of DNA were analyzed using a
melting curve from a real-time PCR instrument (ABI 7900). A mixture
of genomic DNA and PicoGreen melted. Duringcooling theDNA
rehybridizes and PicoGreen intercalates with theDNA.Measured
fluorescenceis relative to the amount of DNA.
34 MOLECULAR BIOLOGY LABORATORY LAYOUT