Molecular Traceability of animals and their products Dr. ZIAD W. JARADAT Department of Biotechnology and Genetic Engineering JUST.

Molecular Traceability of Molecular Traceability of animals and their productsanimals and their products

Dr. ZIAD W. JARADATDr. ZIAD W. JARADATDepartment of Biotechnology and Genetic Engineering

JUST

Adulteration of meat products by Adulteration of meat products by addition of substances of lower value addition of substances of lower value is prohibited for fair trading, consumer is prohibited for fair trading, consumer protection, religious or public health. protection, religious or public health.

Two main methods are used to detect Two main methods are used to detect such malpractice;such malpractice; Immunological MethodsImmunological Methods DNA-based MethodsDNA-based Methods

Immunolgical methods are currently used but have Immunolgical methods are currently used but have drawbacks as they fail to analyze complex food matrices. drawbacks as they fail to analyze complex food matrices. Therefore, it appears that DNA-based methods are more Therefore, it appears that DNA-based methods are more accurate and will be the focus of this Presentation.accurate and will be the focus of this Presentation.

Why do we need to identify the source of the meat species?Why do we need to identify the source of the meat species? In the last two decades several issues has shattered the In the last two decades several issues has shattered the

consumer confidence with the food producers. Of these, consumer confidence with the food producers. Of these, the ban on hormones in food, BSE in Europe and the the ban on hormones in food, BSE in Europe and the

dioxine crises in Switzerland etc.dioxine crises in Switzerland etc.

Genetic TraceabilityGenetic Traceability

A variety of methods are available for an A variety of methods are available for an individual or species identification. These methods individual or species identification. These methods reveal polymorphism or variation that can be used reveal polymorphism or variation that can be used for the identification. The simplest and the oldest for the identification. The simplest and the oldest markers used for this purpose, is using anatomical markers used for this purpose, is using anatomical and physical polymorphisms like;and physical polymorphisms like; Coat colorCoat color Horns Horns Tattoos Tattoos Typing bloodTyping blood

Biochemical polymorphismBiochemical polymorphism

Other methods targeting phenotypic Other methods targeting phenotypic polymorphism by different techniques polymorphism by different techniques such as;such as;

Isoelectric focusing of certain proteinsIsoelectric focusing of certain proteins Agar gel immunodiffusionAgar gel immunodiffusion Counter immunoelectrophoresisCounter immunoelectrophoresis ELISAELISA

Problems with above methodsProblems with above methods Heat treatment might change the conformation of Heat treatment might change the conformation of

the proteins and thus limit the use of these methods the proteins and thus limit the use of these methods that are protein-based.that are protein-based.

Since DNA is the only basis of genetic differences Since DNA is the only basis of genetic differences between distinct organisms, DNA fingerprinting between distinct organisms, DNA fingerprinting seems to be the ultimate method of biological seems to be the ultimate method of biological individualization.individualization.

Studying DNA polymorphismStudying DNA polymorphism Regions of the genome are known to differ between Regions of the genome are known to differ between

individuals. They are tandem polymorphic sites. individuals. They are tandem polymorphic sites. Various methods have been developed for the typing Various methods have been developed for the typing of eukaryotic DNA. The ideal method is supposed to of eukaryotic DNA. The ideal method is supposed to be reproducible, and thus generate reliable data. be reproducible, and thus generate reliable data.

DNA-based markers could be grouped DNA-based markers could be grouped into 2 types;into 2 types;

Clone/sequence based markersClone/sequence based markers Finger print markers.Finger print markers.

The first method requires the isolation of The first method requires the isolation of cloned DNA and determine its sequence. This cloned DNA and determine its sequence. This category include microsatellites, RFLPcategory include microsatellites, RFLP

The second method requires no knowledge of The second method requires no knowledge of the sequence of the polymorphic region or the sequence of the polymorphic region or isolation of a cloned DNA fragment. This isolation of a cloned DNA fragment. This method include method include RAPDRAPD (Random amplified (Random amplified polymorphic DNA), polymorphic DNA), VNTRVNTR (variable number of (variable number of tandem repeats) and tandem repeats) and AFLPAFLP

(amplified fragment length polymorphism).(amplified fragment length polymorphism).

DNA sequencingDNA sequencing Individual identification can be done by Individual identification can be done by

sequencing portion of the individual genome sequencing portion of the individual genome where a difference can be expected to show where a difference can be expected to show up. However, DNA sequencing is not always up. However, DNA sequencing is not always feasible. Therefore, other methods which are feasible. Therefore, other methods which are more feasible can be used instead.more feasible can be used instead.

PCR-RFLP:PCR-RFLP: DNA restriction enzymes recognize DNA restriction enzymes recognize specific sequences in DNA and catalyze specific sequences in DNA and catalyze endonucleolytic cleavage yielding fragments endonucleolytic cleavage yielding fragments of defined length. These fragments are of defined length. These fragments are separated by EF according to their molecular separated by EF according to their molecular size. size.

Differences in fragments will appear due to single Differences in fragments will appear due to single pb polymorphisms or insertions or deletions of pb polymorphisms or insertions or deletions of blocks of DNA thus resulting in loss of cleavage blocks of DNA thus resulting in loss of cleavage site or formation of new one.site or formation of new one.

This will be manifested in different sizes of DNA This will be manifested in different sizes of DNA fragments. Upon using some software for fragments. Upon using some software for comparison, DNA can be typed and animals can comparison, DNA can be typed and animals can be traced.be traced.

ExampleExamplePCR-RFLP analysis of mitochondrial DNAPCR-RFLP analysis of mitochondrial DNA Mitochondrial DNA accumulates about 10 times mutations Mitochondrial DNA accumulates about 10 times mutations

per unit of time as nuclear DNA and has thousands of per unit of time as nuclear DNA and has thousands of copies per cell. copies per cell.

Amplification of mitochondrial DNA segment is a relatively Amplification of mitochondrial DNA segment is a relatively sensitive procedure and the identification of species can sensitive procedure and the identification of species can be based on the mutations in the amplified product.be based on the mutations in the amplified product.

This can be done by RFLP using an enzyme with a This can be done by RFLP using an enzyme with a recognition sequence either abolished or a new one recognition sequence either abolished or a new one created.created.

Species identification using PCR-RFLP of a mitochondrial Species identification using PCR-RFLP of a mitochondrial cytochrome b segment has been well documented and is cytochrome b segment has been well documented and is used for identification of species origin in cheese products used for identification of species origin in cheese products or meat products.or meat products.

MethodMethod

Mitochondrial cytochrome b fragments are Mitochondrial cytochrome b fragments are amplified using DNA purified from amplified using DNA purified from lymphocyte DNA of certain speacies ( e.g lymphocyte DNA of certain speacies ( e.g taurine cattle, water bufffalo, goat and taurine cattle, water bufffalo, goat and sheep) or DNA isolated from mozzarella and sheep) or DNA isolated from mozzarella and feta samples.feta samples.

The product will be a 359 bp fragment.The product will be a 359 bp fragment. There will be different restriction enzymes There will be different restriction enzymes

that can be used for digestion of the product.that can be used for digestion of the product. Based on the cutting site and size, samples Based on the cutting site and size, samples

of different origin will be differentiated.of different origin will be differentiated.

Disadvantages of RFLP: Disadvantages of RFLP:

1. Most RFLPs are only dimorphic (either 1. Most RFLPs are only dimorphic (either the enzyme cut or does not) the enzyme cut or does not)

2. Labor intensive 2. Labor intensive

Repeated DNA sequence polymorphismRepeated DNA sequence polymorphism: : this is also called Simple Sequence Length this is also called Simple Sequence Length Polymorphisms (SSLPs). These are arrays Polymorphisms (SSLPs). These are arrays of repeat sequences that display length of repeat sequences that display length variations i.e different alleles contain variations i.e different alleles contain different numbers of repeat units.different numbers of repeat units.

In this case SSLPs can be multi-allelic as each In this case SSLPs can be multi-allelic as each can have a number of different length can have a number of different length variants. There are two major types of repeat variants. There are two major types of repeat sequences;sequences;

Minisatellites have repeat units up to 100 bp Minisatellites have repeat units up to 100 bp

(mostly 9-30 bp). These are also called variable (mostly 9-30 bp). These are also called variable number of tandem repeats (VNRT)number of tandem repeats (VNRT)

Microsatellites have repeat units from 1 to 6 bp.Microsatellites have repeat units from 1 to 6 bp.

This method was used to carry the first human This method was used to carry the first human DNA fingerprinting.DNA fingerprinting.

How does this method work?How does this method work? DNA probes have to be developed that are DNA probes have to be developed that are

able to detect large number of able to detect large number of minisatellites loci.minisatellites loci.

DNA to be typed will be digested with DNA to be typed will be digested with certain restriction enzymes, certain restriction enzymes, electrophoresed and transferred to nylon electrophoresed and transferred to nylon membranesmembranes

Hybridization of the cut fragments with the Hybridization of the cut fragments with the probes will be done at low stringency probes will be done at low stringency

This will allow us to detect patterns that This will allow us to detect patterns that are unique for unrelated individuals. are unique for unrelated individuals.

These multilocus probes hybridize with a These multilocus probes hybridize with a family of minisatellites sharing the same family of minisatellites sharing the same core sequence, thus producing the multi-core sequence, thus producing the multi-band finger print pattern producing what band finger print pattern producing what is known as a bar code. is known as a bar code.

Random Amplified Polymorphic DNA Random Amplified Polymorphic DNA (RAPD)(RAPD)

The method reveals sequence polymorphism between different

template DNAs based on selective amplification of DNA sequences that are flanked (by chance) by sequences matching an arbitrarily chosen primer. There is a large

number of RAPD primers available commercially, therefore it is

cheep to do this method.

Disadvantages• Depends on exact conditions of PCR for comparisons,

thus it is hard repeat and compare with others work.

DNA finger printing: Application in Meat DNA finger printing: Application in Meat TraceabilityTraceability

The goal: IDENTIFICATION OF THE SPECIES ORIGIN OF MEAT AND PROCESSED MEAT.Genomic DNA probes have been applied for distinction between DNA samples of most species. These probes are generally found in highly repetitive DNA sequences.

Using these repetitive sequences, they succeeded in differentiating meat from distantly related species such as pig and cattle, however, they reported difficulties in differentiating between beef, meat from sheep and meat from goat because of cross hybridization.

This cross hybridization between probe and DNA sequences from closely related species is reduced by addition of unlabelled DNA from the cross hybridizing species.

As for the sensitivity of species differentiation by DNA

hybridization studies have been conducted on mixtures of raw pork and beef. As little as 0,5% raw pork could be detected using total genomic DNA as well as a cloned pig-specific DNA fragment as DNA probe

DisadvantagesDisadvantages laborious, laborious, costly, costly, time consuming andtime consuming and radioactivity use radioactivity use

Alternative methodAlternative methodUsing minisatelit DNA probes; The probes are highly Using minisatelit DNA probes; The probes are highly

specific for species and the complete test is performed specific for species and the complete test is performed within four hours without special equipment.within four hours without special equipment.

How?How? Highly repeated satellite sequences are easily observed Highly repeated satellite sequences are easily observed

following restriction digestion because of their very following restriction digestion because of their very high copy number and their tandem arrangement. high copy number and their tandem arrangement.

Highly repetitive DNA markers have been used for Highly repetitive DNA markers have been used for determining the species origin of animal tissues in determining the species origin of animal tissues in cases of illegal commercialization and poaching of cases of illegal commercialization and poaching of game animals. E.g.game animals. E.g.

DNA from white- tailed deer, moose, and other species was DNA from white- tailed deer, moose, and other species was examined following digestion with 15 restriction enzymes. examined following digestion with 15 restriction enzymes.

Agarose electrophoresis revealed unique banding patterns for Agarose electrophoresis revealed unique banding patterns for each species.each species.

Molecular traceability of animals Molecular traceability of animals and their productsand their products

The polymerase chain reaction discovery opened the The polymerase chain reaction discovery opened the way to many applications particularly in food analysis. way to many applications particularly in food analysis.

Rapid amplification of specific DNA sequences by PCR Rapid amplification of specific DNA sequences by PCR is a method of considerable interest for species is a method of considerable interest for species differentiation due to its: differentiation due to its: simplicity, simplicity, specificity, specificity, low cost andlow cost and high speed.high speed.

Specific Uses of the MethodSpecific Uses of the Method

PCR has been considered for species-specific PCR has been considered for species-specific amplification of Growth Hormone genes in pig, cattle, amplification of Growth Hormone genes in pig, cattle,

sheep and goat.sheep and goat. This test detected pork in fresh or heated meat This test detected pork in fresh or heated meat

mixtures of pork in beef at levels below 2%.mixtures of pork in beef at levels below 2%.

Universal pair of primers amplifying a fragment of the Universal pair of primers amplifying a fragment of the vertebrate mitochondrial cyt b gene were used to vertebrate mitochondrial cyt b gene were used to differentiate between meats from different species. differentiate between meats from different species. They detected restriction fragment length They detected restriction fragment length polymorphisms specific for pig, cattle, wild boar, polymorphisms specific for pig, cattle, wild boar, buffalo, sheep, goat, horse, chicken and turkey.buffalo, sheep, goat, horse, chicken and turkey.

The PCR-RFLP procedure appeared to be a simple The PCR-RFLP procedure appeared to be a simple method that can also quickly analyse exotic method that can also quickly analyse exotic animals and fish because it does not require animals and fish because it does not require preliminary sequencing of the investigated preliminary sequencing of the investigated fragment. fragment.

PCR-RFLP of a conserved region of the PCR-RFLP of a conserved region of the mitochondrial cytochrome b was employed for mitochondrial cytochrome b was employed for identification of flatfish species. The PCR-RFLP is identification of flatfish species. The PCR-RFLP is cheap, can be used with degraded DNA and easy cheap, can be used with degraded DNA and easy method to the point that it is useful for routine method to the point that it is useful for routine

analysis.analysis.

Species identification and particularly Species identification and particularly identification of bovine materials in animal identification of bovine materials in animal feedstuffs is also essential to control a potential feedstuffs is also essential to control a potential source of bovine spongiform encephalopathy. source of bovine spongiform encephalopathy.

A PCR method that allows rapid detection and A PCR method that allows rapid detection and identification of a bovine-specific mitochondrial identification of a bovine-specific mitochondrial DNA sequence has been developed. This method DNA sequence has been developed. This method detects the presence of bovine material at less detects the presence of bovine material at less than 0,125 % in feedstuffs.than 0,125 % in feedstuffs.

The PCR method also detects genetically modified The PCR method also detects genetically modified organisms and foods. organisms and foods.

Thank You and Good Luck