Molecular Traceability of animals and their products

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Molecular Traceability of animals and their
products

• Dr. ZIAD W. JARADAT

Department of Biotechnology and Genetic
Engineering JUST
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• Adulteration of meat products by addition of
  substances of lower value is prohibited for fair
  trading, consumer protection, religious or public
  health.
• Two main methods are used to detect such
  malpractice
• Immunological Methods
• DNA-based Methods

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• Immunolgical methods are currently used but have
  drawbacks as they fail to analyze complex food
  matrices. Therefore, it appears that DNA-based
  methods are more accurate and will be the focus
  of this Presentation.
• Why do we need to identify the source of the meat
  species?
• In the last two decades several issues has
  shattered the consumer confidence with the food
  producers. Of these, the ban on hormones in
  food, BSE in Europe and the dioxine crises in
  Switzerland etc.

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Genetic Traceability

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

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• Biochemical polymorphism
• Other methods targeting phenotypic polymorphism
  by different techniques such as
• Isoelectric focusing of certain proteins
• Agar gel immunodiffusion
• Counter immunoelectrophoresis
• ELISA

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Problems with above methods

• Heat treatment might change the conformation of
  the proteins and thus limit the use of these
  methods that are protein-based.
• Since DNA is the only basis of genetic
  differences between distinct organisms, DNA
  fingerprinting seems to be the ultimate method of
  biological individualization.
• Studying DNA polymorphism
• Regions of the genome are known to differ between
  individuals. They are tandem polymorphic sites.
  Various methods have been developed for the
  typing of eukaryotic DNA. The ideal method is
  supposed to be reproducible, and thus generate
  reliable data.

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• DNA-based markers could be grouped into 2 types
• Clone/sequence based markers
• Finger print markers.

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• The first method requires the isolation of cloned
  DNA and determine its sequence. This category
  include microsatellites, RFLP
• The second method requires no knowledge of the
  sequence of the polymorphic region or isolation
  of a cloned DNA fragment. This method include
  RAPD (Random amplified polymorphic DNA), VNTR
  (variable number of tandem repeats) and AFLP
• (amplified fragment length polymorphism).

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DNA sequencing

• Individual identification can be done by
  sequencing portion of the individual genome where
  a difference can be expected to show up. However,
  DNA sequencing is not always feasible. Therefore,
  other methods which are more feasible can be used
  instead.
• PCR-RFLP DNA restriction enzymes recognize
  specific sequences in DNA and catalyze
  endonucleolytic cleavage yielding fragments of
  defined length. These fragments are separated by
  EF according to their molecular size.

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• Differences in fragments will appear due to
  single pb polymorphisms or insertions or
  deletions of blocks of DNA thus resulting in loss
  of cleavage site or formation of new one.
• This will be manifested in different sizes of DNA
  fragments. Upon using some software for
  comparison, DNA can be typed and animals can be
  traced.

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Example

• PCR-RFLP analysis of mitochondrial DNA
• Mitochondrial DNA accumulates about 10 times
  mutations per unit of time as nuclear DNA and has
  thousands of copies per cell.
• Amplification of mitochondrial DNA segment is a
  relatively sensitive procedure and the
  identification of species can be based on the
  mutations in the amplified product.
• This can be done by RFLP using an enzyme with a
  recognition sequence either abolished or a new
  one created.
• Species identification using PCR-RFLP of a
  mitochondrial cytochrome b segment has been well
  documented and is used for identification of
  species origin in cheese products or meat
  products.

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Method

• Mitochondrial cytochrome b fragments are
  amplified using DNA purified from lymphocyte DNA
  of certain speacies ( e.g taurine cattle, water
  bufffalo, goat and sheep) or DNA isolated from
  mozzarella and feta samples.
• The product will be a 359 bp fragment.
• There will be different restriction enzymes that
  can be used for digestion of the product.
• Based on the cutting site and size, samples of
  different origin will be differentiated.

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• Disadvantages of RFLP
• 1. Most RFLPs are only dimorphic (either the
  enzyme cut or does not)
• 2. Labor intensive
• Repeated DNA sequence polymorphism this is also
  called Simple Sequence Length Polymorphisms
  (SSLPs). These are arrays of repeat sequences
  that display length variations i.e different
  alleles contain different numbers of repeat
  units.

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• In this case SSLPs can be multi-allelic as each
  can have a number of different length variants.
  There are two major types of repeat sequences
• Minisatellites have repeat units up to 100 bp
  (mostly 9-30 bp). These are also called variable
  number of tandem repeats (VNRT)
• Microsatellites have repeat units from 1 to 6 bp.
• This method was used to carry the first human DNA
  fingerprinting.

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• How does this method work?
• DNA probes have to be developed that are able to
  detect large number of minisatellites loci.
• DNA to be typed will be digested with certain
  restriction enzymes, electrophoresed and
  transferred to nylon membranes
• Hybridization of the cut fragments with the
  probes will be done at low stringency
• This will allow us to detect patterns that are
  unique for unrelated individuals.

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

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Random Amplified Polymorphic DNA (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.

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DNA finger printing Application in Meat
Traceability
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.
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• 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

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• Disadvantages
• laborious,
• costly,
• time consuming and
• radioactivity use

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Alternative method

• Using minisatelit DNA probes The probes are
  highly specific for species and the complete test
  is performed within four hours without special
  equipment.
• How?
• Highly repeated satellite sequences are easily
  observed following restriction digestion because
  of their very high copy number and their tandem
  arrangement.
• Highly repetitive DNA markers have been used for
  determining the species origin of animal tissues
  in cases of illegal commercialization and
  poaching of game animals. E.g.
• DNA from white- tailed deer, moose, and other
  species was examined following digestion with 15
  restriction enzymes.
• Agarose electrophoresis revealed unique banding
  patterns for each species.

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Molecular traceability of animals and their
products

• The polymerase chain reaction discovery opened
  the way to many applications particularly in food
  analysis.
• Rapid amplification of specific DNA sequences by
  PCR is a method of considerable interest for
  species differentiation due to its
• simplicity,
• specificity,
• low cost and
• high speed.

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Specific Uses of the Method

• PCR has been considered for species-specific
  amplification of Growth Hormone genes in pig,
  cattle, sheep and goat.
• This test detected pork in fresh or heated meat
  mixtures of pork in beef at levels below 2.
• Universal pair of primers amplifying a fragment
  of the vertebrate mitochondrial cyt b gene were
  used to differentiate between meats from
  different species. They detected restriction
  fragment length polymorphisms specific for pig,
  cattle, wild boar, buffalo, sheep, goat, horse,
  chicken and turkey.

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• The PCR-RFLP procedure appeared to be a simple
  method that can also quickly analyse exotic
  animals and fish because it does not require
  preliminary sequencing of the investigated
  fragment.
• PCR-RFLP of a conserved region of the
  mitochondrial cytochrome b was employed for
  identification of flatfish species. The PCR-RFLP
  is cheap, can be used with degraded DNA and easy
  method to the point that it is useful for routine
  analysis.

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• Species identification and particularly
  identification of bovine materials in animal
  feedstuffs is also essential to control a
  potential source of bovine spongiform
  encephalopathy.
• A PCR method that allows rapid detection and
  identification of a bovine-specific mitochondrial
  DNA sequence has been developed. This method
  detects the presence of bovine material at less
  than 0,125 in feedstuffs.
• The PCR method also detects genetically modified
  organisms and foods.

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Thank You and Good Luck