PART Ia MOLECULAR BIOLOGICAL METHODS: …catalogimages.wiley.com/images/db/pdf/9780470068090.excerpt.pdf · XK02-04 Genisol Maxi-Prep Kit (single preps) www. abgene. com (continued)

PART Ia

MOLECULAR BIOLOGICAL METHODS:TECHNIQUES EXPLAINED

COPYRIG

HTED M

ATERIAL

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 routine DNAanalysis 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, freez–thaw 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 þ 4�C,long-term storage at �20�C. Avoid thawing andfreezing cycles; transport at�20�C; thawing in a waterbath at 37�C directly before DNA extraction.

Liquid blood for RNAextraction

Preserved with specific buffers [e.g., RNAlater solution(content of Qiagen RNA extraction kits)], storage at�80�C; 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 þ 4�C, long-termstorage and transport longer than 24 h at�20�C. 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 þ 4�C, long-term storage �20�C.Avoid thawing and refreezing.

Sperm samples (conservedfor artificialinsemination)

Storage and transport frozen in liquid nitrogen. Iftransported at þ 4�C or �20�C, 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


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)

þNucleoS

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 agarose–polyacrylamide (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 95�C for 15 to 30 s.

2. Annealing step. The reaction mixture is cooled down to 48 to 60�C 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.


to the single-stranded DNA. The optimal temperature at this step depends on themelting temperature of the primers.

3. Elongation step. At 72�C, 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 heating–cooling 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?pid¼260

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.


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.


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.


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 primer–dimmers, 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.


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 10�C 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


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


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, bioM�erieux) 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).


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

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 the manufacturershould 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.


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

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


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 predisposition“flavor” 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 (Bystr€om 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

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.


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 40–70mg/100mg tissuePlant material 1–30mg/100mg tissueRice grains 15–300 ng/ g grainsProcessed food products 0.1–5mg/100mg product

APPLICATIONS 33

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.


labeled probe will be more specific and show fewer background signals than willassays with unlabeled primers and detection with SybrGreen.

Assays specific for mtDNA are more sensitive than assays for genomic DNA. Theycan be used if analysis of mtDNA is necessary (e.g., for sequencing of humanHV I andII regions) (see Chapter 6). For all assays specific for genomic DNA, the limit ofdetection should be 6 pg DNA per reaction for assays specific for single-copy genes(one copy per haploid genome). If the self-developed assay is less sensitive, the PCRconditionsmay not be optimized or the reference DNAwill have a lower concentrationthan expected or measured. If the assay is more sensitive, it may be because thereference DNA has a higher concentration than expected or the assay detects amulticopy gene (more than one copy per haploid genome).

Degradation of the DNA In most real-time PCR assays, fragments of 60 to 100 bpare amplified. For degraded DNA these short fragments can be overrepresented. This

FIGURE 1.22 Quantification of human DNA with a quantifiler kit. Serial dilutions (1:3) ofhuman DNA leads to corresponding differences in Ct values (b) which should be located on astraight line (a) with a regression coefficient as high as 0.99.

TABLE 1.10 Species-Specific Microsatellites

Species Microsatellite Marker Fragment Size (bp)

Horse AHT 4 140–160Cattle TGLA 53 150–180Pig SW 0155 150–160Dog PEZ 1 96–120Cat F85 183–301

APPLICATIONS 35

can lead to failedmicrosatellite or other PCR analysis evenwhen the optimal amount ofDNA was given to the PCR following the results of real-time PCR analysis. Toovercome this problem, species-specific PCR assays can be developed with longerfragments. The size of these fragments should correspond that of analysis by real-timePCR. Comparison of Ct values for shorter (e.g., 100 bp) and longer (e.g., 300 bp)fragments provides a hint of degradation of the DNA in the sample.

Animal Species Determination (Known Species With orWithout Quantification)Animal species determination in processedmaterial or food products can be performedby PCR amplification of mitochondrial DNA followed by sequencing analysis of theproduct (see Chapter 6) or by RFLP. RFLP analysis can be successful only if the DNAdetected is from a species with a known RFLP pattern. In addition, if DNA from onlyone animal is detected by sequencing analysis or RFLP, there is actually no guaranteethat DNA from other species is also present at very low amounts. Low contaminationscan also be detected by real-time PCR. For sequencing analysis or RFLP, PCR primersare used that amplify DNA from almost all mammals. In contrast, for real-time PCR,primers specific for each species have to be used.

Example 1: DNA Analysis of Carcass Meal After DNA extraction, PCR withuniversal primers should be performed to exclude PCR inhibition. In addition, PCRwith primers specific for species that are allowed to be processed as carcass meal(e.g., pig, chicken) and with primers specific for species that are not allowed to beprocessed (e.g., cattle, sheep) is carried out.

Example 2: Analysis of Raw Gelatin Gelatin can be produced from both bovineand porcinematerial. In Europe, gelatin frompig is preferred; in other countries, gelatinfrom pigs cannot be used, for ethical or religious reasons. To determinewhether gelatinis produced from pigs or cattle, a primer pair should be used that amplifies DNA fromboth species with the same efficiency. The primers have to be located in a conservedregion of the mitochondrial genome, with identical sequences for pig and cattle. Inaddition, two probes must be designed, one specific for bovine DNA labeled with onedye (e.g., FAM) and one specific for porcineDNA labeledwith another dye (e.g., VIC).This assay design has the advantage that controls are included. Since DNA extractedfrom gelatin has to be amplified with the primers, a negative result means that aninsufficient amount of DNA was extracted from the gelatin. For pure gelatin samplesone signal (FAM or VIC) is detected; for mixed samples both signals have to beanalyzed. If both signals are detected, the result of the analysis should be interpretedcarefully because how strong the DNAwas degraded during the production process ofthe gelatin cannot be calculated.When analyzing cowmilk products for gelatin, bovineDNA is detected in every case and it is not possible to determine whether the bovineDNAextracted is from the gelatin or from the cells of themilk. Therefore, the detectionof porcine DNA cannot exclude the admixture of bovine gelatin. For milk productsfrom sheep or goat milk, bovine DNA can be analyzed from intentional orunintentional contamination with cow’s milk or from the addition of bovine gelatin.


Example 3: Analysis of Chondroitine Sulfate Chondroitine sulfate can beproduced from raw material of cattle, pigs, chicken, or sharks. With universalprimers for cytochrome b used for species determination, the amplification of DNAfrom cattle and pigs ismore efficient than the amplification ofDNA from chicken.DNAfrom shark is not amplified using these primers. Therefore, universal primers for thedetection of DNA from all shark species have to be designed. Four independent real-time PCR reactions have to be carried out for the analysis. As described for the analysisof gelatin, it cannot be guaranteed that DNAwill be degraded during the production ofchondroitine sulfate from the various raw materials. Therefore, the quantitative resultsof mixed samples have to be interpreted very carefully. Amplified PCR products can beused for sequencing (e.g., to determine shark species, if necessary).

Example 4: Analysis of Duck Liver Pat�e A sample of duck liver pat�e wasdeclared as a mixture of 20% duck meat and liver and 80% porcine meat, liver, and fat.For PCR with universal primers for cytochrome b, amplification from porcine DNA ismore efficient than that from duck DNA. Therefore, in a first sequencing analysis, noDNA from duck can be detected. However, real-time PCR with primers specific forDNA from pig and duck can confirm the ingredients declared.

DNA/RNAContaminationonSurfacesandDNAse/RNAseContaminationFor some applications or for quality assurance, it can be necessary to controlwhether instruments, surfaces, reaction tubes, or used buffers are contaminated withDNA, RNA, DNAse, or RNAse. For the detection of DNAse or RNAse, knownamounts of DNA or RNA are incubated with the suspected buffers or in the tubes at37 to 40�C for 30 to 60min. After this, real-time PCR is performed with theuntreated DNA or RNA as control. If the analysis of incubated DNA or RNA showsa higher Ct value than the control, and the internal process control gives no hint ofinhibition, DNAse or RNAse was present. For the detection of DNA or RNAcontaminations, humid swab samples (in TE or phosphate-buffered saline buffer)have to be collected from surfaces or instruments and the nucleic acids extracted intothe TE buffer. The buffer can also be incubated in the suspected reaction tube orpipette tip. Real-time PCR is performed with universal primers specific for cyto-chrome b, human DNA, or any other DNA/RNA that is identified as a source ofcontamination in the laboratory.

Detection of SNPs and InDelSNPs and InDel (Insertion/deletion) are distributed equally over the total genome ofanimals, plants, fungi, and bacteria. They have very low mutation rates and thereforecan be used for animal or plant species determination, identification of fungi orbacterial strains, proof of identity, and authenticity testing. Detection of SNPs can becarried out on both genomic and mitochondrial DNA.

For high-throughput screening of SNPs, techniques such as MALDI-TOF-MS(matrix-assisted laser desorption–Ionization time-of-flight mass spectroscopy) areused. Examples of projects are population studies/gene diagnostics, clinical studies,analysis of mitochondrial DNA or Y- chromosomal SNPs, and DNA forensics. The

APPLICATIONS 37

advantages of this technique are possible multiplex analyses, competitive costs peranalysis (a few cents per SNP for multiplex analysis), high throughput (production of10,000 data points per hour), and the detection of SNP and InDel in one assay orreaction. The disadvantages are that only the detection of knownmutations is possible,the fixed costs are high, and a complex infrastructure is necessary for analysis.In laboratories with lower throughput, SNP detection for analysis of food or foodproducts can be performed by real-time PCR or sequencing analysis.

Detection of Genetically Modified OrganismsDue to legislative guidelines (e.g., EU Guideline 90/220), it is necessary to carry outqualitative and quantitative detection of genetically modified organisms (GMOs). IfGMOs are imported into the EU, the product has to be declared as a GMO andinformation as to how the GMO can be identified must be available. However, it hasbeen shown in the past that this does not always happen (e.g., import of nondeclaredGMO rice into the EU from the United States in 2006). Since GMO and non-GMOproducts are stored, transported, and processed in the same facilities, contamination ofnon-GMO products by GMO products can occur unintentionally. Therefore, quanti-tative analysis is performed to determine if the GMO content is higher than a definedthreshold. For the quantitative analysis of several GMOs, reference material withdifferent GMO/non-GMO ratios is commercially available. By an analysis of ahousekeeper gene specific for the plant species, the total amount of DNA presentin the single reactions could be determined. PCR is performed in parallel specificallyfor the sequences of the transgenic region that have been published (e.g., a promoter ofcauliflower mosaic virus). From the ratio of the Ct values of the two genes of thereference samples with a known content of GMO and the samples to be analyzed, theGMO content of the samples analyzed can be determined.

De novo detection of GMO is more difficult. If there is any doubt that a samplecontains transgenicDNAsequences, an analysis has to be performed specifically for allknown sequences used for the development of the GMO. This analysis should be donein specialized laboratories. After the new transgenic sequence is detected andpublished, the analysis can also be performed in other laboratories.

REFERENCES

Ballard SA, Grabsch EA, Johnson PDR, GraysonML (2005). Comparison of three PCRprimersets for identification of vanB gene carriage in feces and correlation with carriage ofvancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli. Anti-microb. Agents Chemother., Jan., 49(1):77–81.

Bradbury L, Robert H, Jin Q, Reinke RF, Waters DLE (2005a). A perfect marker for fragrancegenotyping in rice. Mol. Breed., Nov., 16(4):279–283.

Bradbury LMT, Fitzgerald TL, Henry RJ, Jin Q, Waters DLE (2005). The gene for fragrance inrice. Plant Biotechnol. J., 3(3):363–370.

Bystr€om M, B€ocher S, Magnusson A, Prag J, Johansson A (2005). Tularemia in Denmark:identification of a Francisella tularensis subsp. holarctica strain by real-time PCR and


high-resolution typing by multiple-locus variable-number tandem repeat analysis. J. Clin.Microbiol., Oct., 43(10):5355–5358.

Dutka-Malen S, Evers S, Courvalin P (1995). Detection of glycopeptide resistance genotypesand identification to the species level of clinically relevant enterococci by PCR. J. Clin.Microbiol., 33:24–27.

Gallagher ES (1994). A PCR-based sex-determination assay in cattle based on the bovineamelogenin locus. Anim. Genet., Dec. 25(6):425–427.

FSA (Food Standards Agency) (2004). Survey on Basmati Rice. FSIS 47/04. Food safety andInspection service, U.S. Department of Agriculture, Washington, DC.

Gilbert W (1978). Why genes in pieces? Nature, Feb. 9, 271(5645):501.

Griffiths R, Double MC, Orr K, Dawson RJG (1998). A DNA test to sex most birds. Mole.

Ecol., 7(8):1071–1075.

ILAC (International Laboratory Accreditation Cooperation) (2002). Guidelines for ForensicScience Laboratories. ILAC-G19:2002. ILAC, silver water, Australia.

MullisKB (1990). Target amplification forDNAanalysis by the polymerase chain reaction.Ann.Biol. Clin. (Paris), 48(8):579–582.

Qin Y, Liu X, Zhang H, Zhang G, GuoX (2006). Segregation and linkage analysis of 75 novelmicrosatellite DNA markers in pair crosses of Japanese abalone (Haliotis discus hannai)using the 50-tailed primer method. Mar. Biotechnol. (NY), Sept.–Oct. 8(5):453–466.

Schubbert R (2002). Detection ofmicrovariants and large alleles inmicrosatellite systems SE33/ACTBP 2, FGA/FIBRA and D21S11. Presented at the 14th International Symposium on

Human Identification.

UrdiainM, Dom�enech-S�anchez A, Albert�ı S, Bened�ıVJ, Rosselló JA (2004). Identification oftwo additives, locust bean gum (E-410) and guar gum (E-412), in food products by DNA-based methods. Food Addit. Contam., 21(7):619–625.

Walker JA, Hughes DA, Anders BA, Shewale J, Sinha SK, Batzer MA (2003). Quantitativeintra-short interspersed element PCR for species-specific DNA identification. Anal. Bio-chem., May 15, 316(2):259–269.

Zinovieva N, Palma G, M€uller M, Brem G (1995). A rapid sex determination test for bovineblastomeres using allele-specific PCR primers and capillary PCR. Theriogenology, 43:365.

REFERENCES 39

Documents

PART Ia MOLECULAR BIOLOGICAL METHODS: …catalogimages.wiley.com/images/db/pdf/9780470068090.excerpt.pdf · XK02-04 Genisol Maxi-Prep Kit (single preps) www. abgene. com (continued)