Molecular Traceability of animals and their products

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

2. 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

3. 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.

4. 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

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

6. 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.

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

8. 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).

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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

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

21. 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.

22. 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.

23. 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.

24. 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.

25. 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.

26. Thank You and Good Luck