PCR Protocol For Fish and Seafood Authentication MSC Thesis

ERASMUS MUNDUS MASTER COURSE SEFOTECH.
NUT
The Optimization and Validation of a Polymerase Chain Reaction Protocol for Fish and Seafood Authenticity based on the Cytochrome b Gene
M.Sc. thesis submitted by: Dwiyitno
Dwiyitno
Digitally signed by Dwiyitno DN: cn=Dwiyitno, c=ID, ou=SefotechNUT, email=dwiyitno@yahoo.com Date: 2009.04.11 18:47:54 +07'00'
Catholic University of Applied Science (KaHo) Sint Lieven, Belgium Dublin Institute of Technology, Ireland Universidade Catlica Portuguesa, Portugal Anhalt University of Applied Sciences, Germany
2008
KaHo-Sint Lieven School for Engineering Gebr. Desmetstraat 1 9000 Gent - Belgium
The Optimization and Validation of a Polymerase Chain Reaction Protocol for Fish and Seafood Authenticity based on the Cytochrome b Gene
M.Sc. thesis submitted by: Dwiyitno
Project Coordinator: Prof. Dr. Chris Van Keer Supervisors: Prof. Dr. Chris Van Keer Dr. Koen Parmentier Co-supervisor: Stefan Hoffman, M.Sc.
February 2008
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
ABSTRACT
Dwiyitno. The optimization and validation of a polymerase chain reaction protocol for fish and seafood authenticity based on the cytochrome b gene. (Under direction of Prof. Dr. Chris Van Keer and Dr. Koen Parmentier; supervised by Stefan Hoffman, M.Sc) Cytochrome b mtDNA has been widely applied for identification of fish and seafood, either in fresh or processed products. The successful application of product authentication based on genomic profiling considerably depends on the primer design which is used to amplify the targeted DNA fragment. Several primers have been developed specifically to identify particular groups of fish, crustaceans and molluscs. However, universal primers for identification of most fish, crustaceans, and molluscs based on the cyt b region have not been established yet. This study focused on the development of universal primers for fish and seafood authenticity based on the cyt b gene. Universal primers are essential, particularly for identification of unrecognizable samples such as fish fillet, surimi and mixed products. In addition, since DNA quality plays an important contribution on PCR amplification, investigation on the different DNA isolation methods was carried out. Firstly, CytBL1 and CytBH primers which have successfully been used to amplify ~357bp of cyt b gene on various fish were optimized to amplify selected fish, crustaceans and molluscs. Secondly, since this primer couple was not optimum for crustacean, mollusc, and some fishes, degenerate primers were designed by introducing wobbles. Notably, other degenerate primers were evaluated to amplify ~410bp of expected fragment. Evaluation of 3 classical DNA extraction methods and a commercial kit was studied to isolate total DNA of selected samples. The results showed that the CytBL1 and CytBH failed to amplify crustacean and mollusc. Likewise, some species of fish failed to be amplified by this primer couple. The degenerate primers (CytBL1C and CytBHW) are promising to be employed universally for species identification of crustaceans and molluscs. Other universal primers (UCYTB151BF/UCYTB271R and UCYTB152BF/UCYTB271R) effectively amplified all fish, crustaceans, and molluscs tested in this study and thereby can be considered as the first universal primers applicable for fish and seafood. Sequence analysis proved that all validated primers effectively generate 356-358bp and 398-411bp of cyt b region. The similarity index of PCR products against the libraries varied between 92% and 100%. RTPCR was applicable to differentiate between selected samples based on their melting points (Tm). In comparison to the classical methods, the commercial kit offers simplicity procedure and yielded the better quality of DNA isolate for PCR purposes.
Key words: authenticity, mitochondrial cytochrome b, PCR primers, fish and seafood, DNA sequencing
Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)
ACKNOWLEDGEMENTS
This thesis was a part of my master course in Food Science, Technology, and Nutrition (SEFOTECHnut). It was funded by the European Commission under the Erasmus Mundus framework. The studies were undertaken in 2006-2008 at Catholic University of Applied Science (KaHo) Sint Lieven-Belgium as the host university and partially at Dublin Institute of Technology-Ireland, Universidade Cathlica PortuguesaPortugal, and Anhalt University of Applied Sciences-Germany. Many people took part in my M.Sc studies and without them this thesis would not exist. I am deeply grateful to both of my advisors, Prof. Dr. Chris Van Keer (KaHo Sint Lieven) and Dr. Koen Parmentier (ILVO), who have encouraged and mentored me during this works. Prof. Chris is also the coordinator of SEFOTECHnut, thank you for giving me opportunity to be part of this master course. This thesis project was carried out at Institute for Agriculture and Fisheries Research (ILVO), Belgium. I would like to express my gratitude to Stefan Hoffman M.Sc and his research group (Daphne and Sabrine) for the invaluable support and supervising me during this research and writing the report. They have introduced me to many basic theories and practical application in biomolecular work. I gratefully acknowledge Dr. Kris Cooreman, the Director of ILVO-Fisheries Department, for the opportunity to work at his laboratory. Thank all colleagues in Ankerstraat 1 for creating so enjoyable atmosphere to work in. I also thank my reviewers, Prof. Dr. Dirk Iserentant and Prof. Jan Song (Gent University), for evaluating the manuscript and their comments during my public defense. I wish to thank my present employer at Research Center for Marine and Fisheries Product Processing and Biotechnology-Jakarta (Prof. Dr. Hari Eko Irianto, Prof. Dr. Sumpeno Putro, Prof. Dr. Endang Sri Heruwati, and Dr. Singgih Wibowo, M.S.), my former employer (Dr. Ahmad Dimyati, M.S. and Dr. W. Farid Maruf, M.Sc) and all of my workmates, for their support. My warmest thanks belong to my parents, my parents in law, Om Samso-Bulik Murni, Wa Ann and her family in De Pinte, for all the support and understanding. Finally, I deeply thank my beloved wife, Lusi, and my juniors (Rizan and Fitri) for all their patience and comfort during these years.
Gent-Oostende, February 2008 Dwiyitno
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TABLE OF CONTENTS
ABSTRACT ..................................................................................................................... i ACKNOWLEDGEMENTS ............................................................................................. ii TABLE OF CONTENTS ................................................................................................. iii LIST OF FIGURES .......................................................................................................... v LIST OF TABLES ........................................................................................................... vii I. INTRODUCTION .......................................................................................................... 1 II. REVIEW OF LITERATURE ...................................................................................... 3 2.1. The importance of product authenticity ................................................................ 3 2.2. Analytical methods for product identification ...................................................... 4 2.2.1. Traditional approaches ............................................................................... 5 2.2.2. Protein based methods (Proteomics) .......................................................... 5 2.2.3. DNA based methods (Genomics) ............................................................... 6 2.2.4. Other methods ............................................................................................ 8 2.3. Genomic identification based on mitochondrial DNA ......................................... 8 2.4. Fish and seafood authenticity based on the cytochrome b gene ........................... 11 2.4.1. Specimen and treatment of sample ............................................................. 11 2.4.2. Isolation of DNA ........................................................................................ 12 2.4.2.1. Tissue digestion ............................................................................. 13 2.4.2.2. Separating proteins and contaminants ........................................... 14 2.4.2.3. Precipitation and recovery of DNA ............................................... 15 2.4.2.4. Commercial kits ............................................................................. 16 2.4.3. Determination of DNA yield and purity ..................................................... 17 2.4.4. Polymerase chain reaction (PCR) ............................................................... 19 2.4.4.1. Primer design ................................................................................. 20 2.4.4.2. Components of PCR reaction ........................................................ 22 2.4.5. PCR product analysis ................................................................................. 23 2.4.5.1. DNA sequencing .......................................................................... 24 2.4.5.2. Fingerprinting techniques .............................................................. 25 2.4.5.3. Other techniques ............................................................................ 27 III. OBJECTIVES OF PRESENT STUDY ...................................................................... 28 3.1. Problems .............................................................................................................. 28 3.2. Objectives ............................................................................................................ 28 3.3. Research framework ............................................................................................ 29 IV. MATERIALS AND METHODS ............................................................................... 30 4.1. Construction of reference database of cyt b genes .............................................. 30 4.2. Sample collection and preservation ..................................................................... 30 4.3. Comparison of DNA extraction methods ............................................................ 31 4.3.1. DNA extraction method 1 (Promega) ......................................................... 32 4.3.2. DNA extraction method 2 (Hsieh et al., 2005)............................................ 32 4.3.3. DNA extraction method 3 (Wasko et al., 2003) .......................................... 32 4.3.4. DNA extraction method 4 (Asahida et al., 1996) ........................................ 33
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4.3.5. Determination of DNA yield ..................................................................... 33 4.3.5.1. Standard curve .............................................................................. 34 4.3.5.2. Sample Analysis ........................................................................... 35 4.3.6. Evaluation of DNA purity ......................................................................... 35 4.3.7. PCR assay .................................................................................................. 36 4.3.8. Visualization of PCR product .................................................................... 37 4.4. Optimization and validation of a universal PCR protocol ................................... 37 4.4.1. Primer design ............................................................................................. 37 4.4.2. PCR assay .................................................................................................. 38 4.4.3. Direct sequencing of PCR product ............................................................ 38 4.4.4. Real Time PCR application ....................................................................... 39 4.5. Data analysis ........................................................................................................ 40 V. RESULTS .................................................................................................................... 41 5.1. Comparison of DNA extraction methods ............................................................ 41 5.2. Evaluation and optimization of CytBL1 and CytBH primers ............................. 42 5.3. Validation of degenerate primers based on CytBL1 and CytBH ......................... 44 5.4. Optimization of universal primers based on UCYTB151F and UCYTB270R .... 45 5.4.1. Validation of UCYTB151BF/UCYTB271R primers ................................ 46 5.4.2. Validation of UCYTB152BF/UCYTB271R primers ................................ 48 5.5. PCR product analysis .......................................................................................... 50 5.5.1. Direct sequencing ...................................................................................... 50 5.5.2. Melting temperature profiles ..................................................................... 51 VI. DISCUSSION ............................................................................................................ 53 6.1. Comparison of DNA extraction methods ............................................................ 53 6.2. Optimization and validation of universal primers ............................................... 54 6.3. PCR product analysis .......................................................................................... 56 VII. CONCLUSIONS ....................................................................................................... 58 REFERENCES ................................................................................................................. 59 LIST OF ABBREVIATIONS ........................................................................................... 68 LIST OF WEBSITES ....................................................................................................... 60 APPENDIXES .................................................................................................................. 70
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LIST OF FIGURES
II. REVIEW OF LITERATURE Figure 2-1. The application of traceability in fish and seafood product ................................. 4 Figure 2-2. Link between different identification techniques and their tools ........................ 7 Figure 2-3. Illustration of mitochondria in a cell and its genomic map ................................. 10 Figure 2-4. DNA spectra presented by spectrophotometer and spectrofluorometer ................ 18 Figure 2-5. The principle of PCR amplification ..................................................................... 19 III. OBJECTIVES OF PRESENT STUDY ............................................................................ 28 Figure 3-1. The framework of the study ................................................................................. 29 IV. MATERIALS AND METHODS Figure 4-1. Genomic database obtained from NCBI and FishTrace ...................................... 30 Figure 4-2. An example of standard curve for spectrofluorometric measurement .................. 34 Figure 4-3. The application of NanoDrop and its typical data output ..................................... 35 Figure 4-4. Apparatus for PCR application ............................................................................. 36 Figure 4-5. The scheme of targeted fragments generated by the evaluated primers ............... 38 Figure 4-6. Software for sequence chromatogram analysis: ChromasPro and BioEdit .......... 39 Figure 4-7. Typical outputs of RT-PCR: annealing curve and melting curve ......................... 40 Figure 4-8. An example of BLAST analysis obtained via GenBank and FishTrace................ 40 V. RESULTS Figure 5-1. Electrophoresis profile of DNA isolates .............................................................. 42 Figure 5-2. Electrophoresis profile of PCR products following different extraction methods ................................................................................................................................ 42 Figure 5-3. Electrophoresis profile of PCR products with CytBL1/CytBH on fish ................. 43 Figure 5-4. Electrophoresis profile of PCR products with CytBL1/CytBH on crustaceans and molluscs ................................................................................. 43 Figure 5-5. Electrophoresis profile of PCR products at different concentrations of MgCl2 ... 43 Figure 5-6. Electrophoresis profile of PCR products produced by degenerate primers based on CuyBL1/CytBH on selected samples ................................................... 44 Figure 5-7. Electrophoresis profile of PCR products with CytBL1C/CytBHW on fish .......... 44 Figure 5-8. Electrophoresis profile of PCR products with CytBL1C/CytBHW on crustaceans ................................................................................................................. 45 Figure 5-9. Electrophoresis profile of PCR products with CytBL1C/CytBHW on molluscs 45
Figure 5-10. Electrophoresis profile of PCR products produced by UCYTB151BF on selected samples ............................................................................................ 45 Figure 5-11. Electrophoresis profile of PCR products produced by UCYTB152BF on selected samples ............................................................................................ 46 Figure 5-12. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-A ............................................................................................................. 46 Figure 5-13. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-B ....................................................................................................................... 47 Figure 5-14. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on crustaceans ............................................................................................................. 47 Figure 5-15. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on molluscs .................................................................................................................. 47 Figure 5-16. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-A ...................................................................................................................... 48 Figure 5-17. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-B ....................................................................................................................... 48 Figure 5-18. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on crustaceans ............................................................................................................. 49 Figure 5-19. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on molluscs .................................................................................................................. 49 Figure 5-20. Variations of melting peak on selected samples ................................................. 52 VI. DISCUSSION Figure 6-1. Multiple alignment of evaluated primers with cyt b gene of selected references.. 55
APPENDIXES Figure I. List of samples used in this study ......................................................................... 70
Figure III. Electrophoresis profile of gradient tests ............................................................... 72 Figure IV. Sequence chromatogram profiles of PCR products ............................................. 74
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LIST OF TABLES
II. REVIEW OF LITERATURE Table 2-1. Applicable categorical levels of each molecular marker or gene region ............... 10 Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood ............. 21 IV. MATERIALS AND METHODS Table 4-1. Protocol used to prepare the standard curve ......................................................... 34 Table 4-2. Composition of PCR reaction ............................................................................... 36 Table 4-3. Set of primers used in this study ........................................................................... 37 Table 4-4. PCR condition of the different primer sets ........................................................... 38 Table 4-5. Reaction components of RT-PCR amplification ................................................... 39 Table 4-6. The thermal profile of RT-PCR amplification ...................................................... 40 V. RESULTS Table 5-1. DNA concentration and purity yielded by different extraction methods ............... 41 Table 5-2. Optimal annealing temperature (C) of validated primer couples .......................... 49 Table 5-3. Sequence analysis of selected samples .................................................................. 51 Table 5-4. Variations of melting point and GC content of selected samples .......................... 52
APPENDIXES Table II. List of chemicals, solution and equipment ............................................................ 71
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CHAPTER I INTRODUCTION
Authenticity of fish and seafood products is important to implement the labeling regulation as set by many countries. The EU, for example, regulates traceability of feed and food ingredients and food sources to protect consumers from fraud and adulteration. Identification of product authenticity is essential to prevent substitution of commercially important products with less valuable species (Trotta et al., 2005). In addition, authenticity is advantageous for either food producers or food inspectors to assure the species included in the formulations of the products (Pineiro et al., 1999). Recently, traceability is also becoming a concern in Canada, Japan, Australia, and New Zealand (Loftus, 2005; Food Quality News, 2006). Traditionally, identification of fish is carried out based on the available external characteristics. With concern to the prospect of growing international trade and an increasing number of potentially marketable species, it is worthwhile to conduct rapid and accurate analytical methods to distinguish fish and seafood products without using morphological features. Protein electrophoresis and molecular biological methods are valuable methodologies for identification of fish and seafood authenticity (Bossier, 1999; Etienne et al., 1999; Rehbein et al., 1999; Pineiro et al., 1999; Bossier & Cooreman, 2000). Protein electrophoresis is an earlier method developed using either SDS-PAGE, agarose GE or IEF patterns of water soluble sarcoplasmatic protein. Nevertheless, there are disadvantages regarding these identification methods. As protein is denatured or altered in consequent of processing, especially by heating, protein based techniques are only applicable to fresh/raw products, frozen fillets and mildly treated fish and seafood (Wolf et al., 2000). In addition, the resulted patterns from these methods can be different depending on the individual age, length, development stage, or environment condition which in turn leads to a high degree of intra species variation. The most prominent techniques for species identification are based on the analysis of nucleic acids, either nuclear or mitochondrial DNA (Pineiro et al., 1999). The significant advantage of DNA based methods is their ability to identify not only fresh and frozen, but also processed, degraded and mixed products. In general, DNA based techniques rely on polymerase chain reaction (PCR) and PCR product analysis. Fingerprinting techniques, for
instance RFLP, AFLP, RAPD, SSCP, and DGGE/TGGE, have been demonstrated as fast, cheap and straightforward gene identification (Mackie et al., 2000; Hird et al., 2005). However, sequencing is known as the most accurate technique to produce a high resolution genomic identity among examined species (Bartlett & Davidson, 1991; Unseld et al., 1995). Mitochondrial DNA (mtDNA) has been used world wide for species identification, including fish and seafood products. Since mtDNA is maternally inherited, the use of this DNA may reduce the taxonomic uncertainty caused by hybridization which exists in nDNA (Ward et al., 2005). Specifically, DNA barcodes focus on the 600 bp sequence of cytochrome oxidase I (COI), while the EU has been developing genomic data base for commercially important fish based on mitochondrial cytochrome b (cyt b) and nuclear rhodopsin (Ratnasingham & Hebert, 2007; www.fishtrace.org). Cyt b gene has been widely applied to identify various species of fish and seafood. Several protocols have been developed specifically to identify particular groups of fish and seafood, including tunas/family Scombroidae (Bartlett & Davidson, 1991; Rehbein et al., 1997), caviar (DeSalle & Birstein, 1996), salmonoid (Rehbein et al., 1997), flatfish (Cspedes et al., 1998), mollusc and arthropods (Meritt et al., 1998), puffer fish (Hsieh et al., 2003), gadoid/family Gadidae (Aranishi et al., 2005), billfish/family Istiophoridae (Hsieh et al., 2005; Richardson et al., 2006) and the most recently teleost fish (Sevilla et al., 2007). However, a universal protocol, particularly universal primers based on cyt b gene for identification of the majority of fish, crustaceans, and molluscs, has not been established yet.
CHAPTER II REVIEW OF LITERATURE

2.1. The importance of product authenticity Traceability is increasingly recognized as a standard management tool across the agri-food industry, mainly due to recent trends on food crises as well as the consequent demands for global transparency within the food chain (Loftus, 2005). Studies in the U.S. fish market showed that due to the highly demand and popularity, at least 77% of red snapper were commonly mislabeled or substituted with the less valuable species (Marco et al., 2004). Similarly, the expensive category of grouper (genera Epinephelus and Mycoteroperca) are frequently misidentified as nile perch (Lates niloticus) or the wreck fish (Polyprion americanus), which is closely related and can not be identified when their morphological features disappeared (Trotta et al., 2005). This mislabeling, misbranding, and economic adulteration are illegal practices and consequently to gain financially consumer fraud. From the viewpoint of food safety, authenticity could protect public from poisoning incidents due to ingestion of toxic products. Quality assurance has become the top priority of meat retailers and producers due to the Bovine Spongiform Encephalopathy (BSE) crisis. BSE almost caused the collapse of beef industry in England and the rest of Europe, when their beef was banned in many countries (Hanluain, 2001; Necidov et al., 2002). Serious food poisoning incidents due to ingestion of toxic puffer fish or toxic dried fish fillets have been reported in Taiwan (Hwang et al., 1995; Hsieh et al., 2002, Cheng et al., 2003). This is due to the difficulty for manufacturers and consumers to distinguish morphological similarities between Lagocephalus lunaris and L. gloveri. Additionally, Takifugu oblongus is often abused as the material for dried fish fillet. With respect to the fact that awareness on GMOs food arises all over the world, it becomes essential to establish consumer protection from GMO jeopardy (Reg. No.1829/2003). For traceability along the food (supply) chain, particularly for meat and meat products, several aspects, such as information of animal species, origin, age, composition and production system are important (Schwagele, 2005). Regulation (EC) 2065/2001 for example, emphasizes that traceability of fish product requires information concerning species identity, geographical origin, and method of production (wild or farmed, organic or intensive). Figure 2-1 shows the illustration of DNA application on the traceability of
seafood product. Hence, for large scale biodiversity monitoring purposes, it is worthwhile to develop a rapid and accurate identification tool. Interestingly, deoxyribonucleic acid (DNA) based technology can overcome the difficulties of tracing animal identity and animal by-products shown by conventional tagging and labelling systems (Hayes et al., 2005).
RFID movement monitoring (Growing stage)
Barcode label tracking (Processing & distribution)
RFID & Hatchery sampling
DNA identity
Harvest sampling
DNA identity
Retail sampling
Figure 2-1. The application of traceability in fish and seafood product (Re-illustrated based on Loftus & Laronde, 2005; Thompson et al., 2005)
2.2. Analytical methods for product identification A key feature of any traceability system has to be able to clearly identify that to be traced. Ideally, the product identifier should: (a) uniquely identify the unit or batch, (b) retain identity throughout the product life-cycle, (c) not hinder its host, and should be (d) secured (fraud proof) and (e) permanent, (f) simple to read and capture identifying data (Loftus, 2005). Accordingly, there is no single identification system likely to meet all of these requirements. Generally, two types of product identifier can be distinguished, i.e. external identifiers and internal/biometric identifiers that are an integral part of the animal or product. External types include either manual methods such as paper labels, ink brands (tattoos), and ear tags, or electronic methods such as Radio Frequency Identification (RFID) tags and injectable microchips (Loftus & Laronde, 2005; Thompson et al., 2005). The advantage of these approaches is their ability to encode different types of information and the relative ease of reading the data, particularly from electronic identifiers. On the other hand, biometric labelling systems incorporate biological data and therefore cannot easily be faked, altered or appropriated. The technologies include DNA profiling, retinal scanning and nose printing (Loftus & Laronde, 2005).
2.2.1. Traditional approaches The quality of food products can be determined by combining sensory analysis with specific microbiological and chemical tests (Suvanich et al., 2000). However, sensory profiles between the closely-related species are difficult to be characterized by a consumer panel even if a trained. Recently, an integration of electronic tongues and noses is used to describe the identification and classification based on flavor and aroma and other measurements of quality (Deisingh et al., 2004; Gomez et al., 2007). The techniques rely on the information obtained from the multi-sensor arrays as principal components analysis and artificial neural networks. The chemical composition and nutritive value such as moisture, crude protein, ash, and fat can significantly distinguish the different species of certain fish and seafood (Hong, 1991; Celik et al., 2004). A study conducted on crabs by Tureli et al. (2000) for example, revealed that the ratio of crude protein, dry matter, and crude ash in the meat of male crabs was higher in swimmer crabs (Portunus pelagicus) than in blue crabs (Callinectes sapidus). A different study revealed that significant differences existed in the protein contents of claw and body meat between those two species (Gokoolu & Yerlikaya, 2003). Since fatty acid composition is practically a reflection of the diet, its profile can be used to discriminate wild from cultivated fish (Martinez et al., 2003; Moretti et al., 2003). Nonetheless, composition and nutritive value may differ depending on region, habitat, sex, age, stages of life or sexual cycles, seasonal factors, and diet (Tsai et al., 1984; Reddy et al., 1991). 2.2.2. Protein based methods (Proteomics) Proteomics emerged in the beginning of the 1990s due to the need for new protein analysis methods. The proteome is defined as the entire protein complement expressed by a cell type of an organism (Wilkins et al., 1996). Proteome research focuses on the structural and functional analysis of the proteome and the interaction of proteins. This includes the isolation, identification and characterization of all proteins expressed by the organisms genome. Proteome analysis could lead the way to explain the function of an organism dynamically rather than statically. This is important since the protein compositions and concentrations change from cell type to cell type, even within sub-cellular compartments. Moreover, they differ between various stages of development (Ferguson et al., 1995).
Proteins (enzymes, myoglobin, etc.) have been widely used as species markers. Applicable techniques include separation of water-soluble proteins by starch, polyacrylamide or agarose gel electrophoresis, IEF, and two-dimensional (2D) electrophoresis (OFarrell, 1975; Pineiro et al., 1999; Martinez et al., 2005 & 2007). Highly resolved water-soluble protein patterns can be used to differentiate genetically close-related species. Interestingly, the limit of detection of gel electrophoretical methods varies between 0.1% and 1% (Xie, 2003). Notably, proteomics can be used to differentiate species, breeds, and varieties by their specific protein pattern. One and 2D protein electrophoresis are the most frequently used techniques for proteome research, while peptide mass fingerprinting analysis by MALDI-TOF mass spectrometry has become the most powerful techniques for proteome analysis (Boucherie et al., 1995; Xie, 2003). Immunological techniques, like ELISA, performed on the solid surface of microwell plates are using suitable target proteins for analysis. A qualitative detection of animal species is possible and the limit of detection depends upon their content in meat products, for instance in pork 61%; poultry and beef 62%; sheep 65% (Schwagele, 2005). Other protein separation and quantification techniques include liquid chromatography (LC/HPLC) and capillary electrophoresis (Lpez, 2007). Nevertheless, electrophoresis has some drawback to identify processed products. Variation in factors, such as temperature, pasteurization, and processing parameters, affect the results (Rehbein, 1990; Barlett et al., 1993; Xie, 2003). In addition, immunological methods may generate cross-reaction among protein from closely related species (Wolf et al., 2000; Necidov et al., 2002).
2.2.3. DNA based methods (Genomics) The field of genomics utilizes a variety of technologies to study the information content of cells. The genome research generally refers to sequencing the total genomic DNA of an organism and mapping all genes within these sequences (Brooker, 1999). In contrast to the proteome, genomic research focuses on the structural and functional analysis of the gene as well as their recombination. This includes the isolation, identification and characterization, either physical or genomic mapping, of the entire genome (Johnson & Browman, 2007). Historically, the term genome was emerging in 1970s with the introduction of techniques for manipulating DNA (recombinant) and reading its sequence (Watson & Berry, 2003).
DNA-based techniques have been demonstrated as fast, cheap and straightforward gene identification (Mackie et al., 1999; Weder et al., 2001). Using DNA has many advantages as it is unique to the individual and creates a means of permanent identification throughout the life cycle of the animals. DNA traceability infrastructure could also be used to check for the presence of a particular genetic modification (Loftus, 2005). In some European markets, a routine program of verification sampling and DNA analysis/matching is used to monitor the ability of a supply chain to provide traceable products. The significant advantage of DNA-based methods is the applicability for identification of not only fresh and frozen products, but also processed, degraded and mixtures, which are not well achieved by protein-based method. Figure 2-2 reveals the link between different identification techniques and their tools (Dupont et al., 2007).
Metagenomics Community Species Genomics Transcriptomics Proteomics DNA mRNA Protein ESTs Microarrays Toolbox Sequencing Barcoding
1D & 2D Gel Electrophoresis
Figure 2-2. Link between different identification techniques and their tools (Dupont et al., 2007) ESTs: expressed sequence tags In industrial scale, DNA based traceability has been applied on fresh beef in the UK and Ireland since the late 1990s (Hanluain, 2001). Recently, DNA barcoding has globally gained much attention. Founded in 2004 at University of Guelph (Ontario), Barcode of Life Data System (BOLD) establishes an integrated platform for species identification based on DNA profile. The concept of DNA barcoding has been applied on North American bird species (Hebert et al., 2004), Australian fish (Ward et al., 2005), and a variety of other invertebrate (Barrett & Hebert, 2005; Smith et al., 2005). Development of DNA barcoding systems will make large-scale applications of DNA traceability increasingly cost effective and feasible.
2.2.4. Other methods Visible and Near-Infrared (VIS/NIR) spectroscopy has been used to detect adulteration in many high-value foods. VIS/NIR spectroscopy is an affordable, powerful, and objective tool that requires minimal sample preparation and can creates fast results. In addition, it does not require highly trained people to operate the instrument and interpret the results. Few biological compounds absorb light in the visible region (380 700 nm), which is usually function-related absorption (Pasquini, 2003). In contrast, biochemical compounds such as DNA and RNA absorb light in the near ultraviolet range, though absorption is unrelated to function. Absorption in the near-infrared region (780-2500 nm) arises from vibration motion of hydrogen molecules (Gayo, 2006). VIS/NIR technology has been used to determine the authenticity of olive oil that was adulterated by other vegetable oils (Tay et al., 2002) and adulterated honey by sugar solutions (Kelly et al., 2004). In fishery products, VIS/NIR spectroscopy has been used to detect economic adulteration of Atlantic blue and blue swimmer crab meat which is adulterated with surimi-based imitation crab meat (Gayo, 2006). Similarly, nuclear magnetic resonance (NMR) can be used to obtain lipid fingerprints. By using this technique, Martinez et al. (2003) revealed some nutraceutical fish oil capsules were incorrectly labeled concerning the species and the lipid content. The variations of isotopic abundances, such as hydrogen, carbon, nitrogen, and oxygen, are of particular interest for food authentication studies. Since the variation of isotopic abundance in animals is mainly due to the origin, nature of the feed, and animals metabolism, it can be used both for origin assignment and to detect adulteration. The isotope ratio of C and N has been used to examine the authentication in wild and farmed Atlantic salmon (Dempson & Power, 2004) and European sea bass/Dicentrarchus labrax (Sweeting et al., 2007). 2.3. Genomic identification based on mitochondrial DNA Mitochondria are essential organelles in the cytoplasm since they serve many important functions for the cell especially oxidative ATP-production, degradation of fatty acids, and modulation of intracellular calcium homeostasis. They also play a major role in cell signaling and apoptosis, as well as in biosynthesis (e.g. heme-groups, nucleotides, and amino acids) and degradation (e.g. urea cycle) of metabolites (Kleinsmith & Kish, 1995). ATP production is regulated in the citric acid cycle, also known as Krebs cycle. This is the final common pathway for different metabolites such as carbohydrates, fatty acids and
amino acids. The compounds with a high redox-potential (reduced nicotinamide-adeninedinucleotide/NADH) and reduced flavin-adenine-dinucleotide (FADH2) are generated in this cycle. The products are later delivered to the respiratory chain of the mitochondrion in order to generate ATP. Mitochondria are comprised of two membrane systems: intra (inner) and extra (outer) membranes. In the center of the mitochondrion and between the membranes there are two aqueous compartments: the matrix and the inter-membrane space (Kleinsmith & Kish, 1995). The two membrane systems contain carrier proteins and channels that regulate the exchange of substrates between the compartments. The inner membrane is typically rich in proteins in which the high molecular weight multi-protein-complexes of the respiratory chain are located at the inner mitochondrial membrane. The total number of different proteins or polypeptides making up a mitochondrion is estimated to be around 1000 (Kleinsmith & Kish, 1995). Basically, a genetic profile or molecular identity can be provided from either nuclear (nDNA) or mitochondrial genome (mtDNA). MtDNA has been successfully used as a biological marker (Avise et al., 1987; Esposti et al., 1993; Lin et al., 2005) and has many advantages: 1. MtDNA is unique as it is maternally inherited in most species. This means that only the mtDNA of the oocyte is transmitted to the offspring. Exceptions with paternal leakage exist in mice and double uniparental inheritance in sea mussels (Mytilidae), freshwater mussel (Unionidae) and clam (Veneridae) (Hoeh et al., 1991; Burzynski et al., 2006). 2. MtDNA has a high mutation rate since it is vulnerable due to its compact structure, lack of histone protection, insufficient repair mechanisms and exposure to reactive oxygen species generated along the respiratory chain. This vulnerability results in the higher mutation rate than the nuclear DNA (Zeviani et al., 1998). 3. The high copy number of mtDNA facilitates a successful analysis of degraded or very limited amount of raw material (Watson & Berry, 2003). 4. Integrated genomic database of mtDNA has been established by various institutions, such as NCBI, BOLD, CBOLs, FISH-BOL, and Fishtrace. MtDNA is a small circular molecule (Figure 2-3), generally between 14 and 18 kb with exception in certain individual, such as sea scallop (family Pectenidae) which contains up to +35 kb (Snyder et al., 1987). Typically, mtDNA is composed of 37 genes coding for 22 tRNAs, 2 rRNAs (12S and 16S) and 13 mRNAs coding for proteins. The
mitochondrial genome is arranged very efficiently as it lacks introns and has small intergenic spacers where the reading frames sometimes overlap. The control region is the primary non-coding region, and is responsible for the regulation of heavy (H) and light (L) strand transcription and of H-strand replication (Kleinsmith & Kish, 1995).
Control region (D-Loop) Cyt b 12SrRNA ND6 Mitochondria Nucleus 16SrRNA 22 tRNA genes
12 protein-coding regions
ND5
ND1 ND2
ND4
ND4L ND3 COIII ATPase6 ATPase8
COI COII
Figure 2-3. Illustration of mitochondria in a cell (left) and its genomic map (right) Table 2-1. Applicable categorical levels of each molecular marker or gene region
Kingdom A. Nuclear DNA 1. SSU (16-18S) 2. LSU (23-28S) 3. 5.8S 4. IGS 5. ITS 6. Rhodopsin*) B. Mitochondrial DNA 1. Ribosomal RNA - 12SrRNA - 16SrRNA 2. Protein 3. Coding genes - ND1 - ND2 - COI**) - COII - Cyt b*) 4. Control region Phylum Class Order Family Genus Species
+++++++++++++++++++++++++++-----++++++++++++++++++------+++++++++++++++++++++-----+++++ +++++++++++
+++++++++++++++++++-----+++++++++++++-----
-------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ +++++
The bold lines indicate as mostly applicable categorical levels for each molecular marker or gene region, while the dot lines indicate less frequently applicable
*)
: FishTrace;
**)
: Barcodinglife
10
Analysis of mtDNA is commonly accomplished by sequencing or fingerprinting techniques of particular coding regions. Ribosomal RNA (12SrRNA and 16SrRNA), cytochrome oxidase, cytochrome b, and D-loop or control region are commonly employed for species identification (Passarino et al., 2002; Scander & Halanych, 2003; Watanabe et al., 2004; Barlaan et al., 2005; Trotta et al., 2005; Goldenberg et al., 2007). Table 2-1 describes the applicable categorical levels of each molecular marker or gene region for species identification (Hwang & Kim, 1999; FishTrace; Barcodinglife). In recent time, mtDNA has been widely used for species identification of fish and seafood products which are mostly focused on COI and cyt b regions (Ward et al., 2005; Ratnasingham & Hebert, 2007; Roe & Sperling 2007; Sevilla et al., 2007). 2.4. Fish and seafood authenticity based on the cytochrome b gene Cytochrome b is one of the cytochromes involved in the electron transport in respiratory chain of mitochondria. Located between tRNA-Glu and tRNA-Thr, cyt b contains eight transmembrane helices connected by intra membrane or extra membrane domains. The combination of cyt b gene and other genes in genomic DNA encodes for cytochrome c oxidoreductase, which is a complex enzyme in oxidative phosphorylation (Kleinsmith & Kish, 1995). Cyt b gene as a phylogenetic probe is widely used because it is easier to align a protein-coding sequence that has evolved over the period than to align either mitochondrial rDNA or noncoding sequences (Irwin et al., 1991). Kocher et al. (1989) have shown that some highly conserved regions on the mitochondrial cyt b gene are suitable for species identification in most vertebrate species. The wide use in phylogenetic study has resulted cyt b as a universal marker. So far, cyt b has been the most prevalent source of sequence data in fish (Bartlett & Davidson, 1991; DeSalle & Birstein, 1996; Rehbein et al., 1997; Cspedes et al., 1998; Hsieh et al., 2007; Sevilla et al., 2007). However, the successful application of genomic based traceability heavily depends on DNA sampling and DNA analysis. Generally, DNA based identification mainly relies on the application of PCR to generate desired fragment of specific region of DNA template. 2.4.1. Specimen and treatment of sample DNA isolation is one of the important parts in PCR application since it is used as template for PCR amplification. The DNA sampling method should provide high-quality
11
and high-quantity DNA useful to accommodate the targeted region. Basically, DNA isolate can be obtained from any biological material. Technically, DNA sampling must be lowcost, relatively easy to perform and produce samples in a format suitable for laboratory analysis (Loftus & Laronde, 2005). However, non-destructive DNA isolation is desirable, especially when working with live samples, or with threatened or endangered species, where sacrificing the animals is unaffordable (Wasko et al., 2003). There have been a number of innovations in DNA sampling specimen conservation. Muscle tissue (mostly white muscle) represents the most common used sources of fish DNA. Meanwhile, egg, liver and blood are used when the individuals are too small to be effectively sampled by muscle (Barlett & Davidson, 1992; Martinez et al., 1998; Watanabe et al., 2004). Moreover, sampling strategy for non-destructive DNA isolation can be achieved from fish fins or scales (Wasko et al., 2003). Lysis buffer may be used for long periods of delay before the specimen is extracted. The most used lysis buffer for specimen preservation contains Tris-HCl, NaCl, EDTA, and SDS (TNES) either with or without urea (4 to 8 M). In addition, formaldehyde has been recognized for fixing and preserving biological specimens, especially for museum collection. These preserving materials have been used to store fin and scale samples of fish for years with good DNA quality (Asahida et al., 1996; Schander & Halanych, 2003). 2.4.2. Isolation of DNA The isolation of highly-quality DNA is the major step for various molecular-biology techniques. Low amount of DNA with the presence of PCR inhibitors are common factors to compromise the PCR amplification (Weder, 2002; Di Pinto et al., 2007). Proteins, RNAs, lipids, polysaccharides, and other leftover cellular constituents, are frequently present in the DNA, particularly when isolated by classical methods. During PCR amplification, these contaminants may interfere with restriction enzymes, ligases, and thermostable DNA polymerase (Merente et al., 1998). Rationally, DNA isolation procedures should aim to solubilize cellular components and simultaneously inactivate any intracellular nucleases to conserve biologically active DNA. Most isolation procedures combine the use of one or more agents, such as organic solvents, detergents, N-lauryl sarcocyl, chaotropic salts, urea, etc. Occasionally, mercaptoethanol is added for protein denaturation (Merente et al., 1998). Recently,
12
numerous commercial kits are available for isolation of total DNA. The use of commercial kit permits production of higher quality DNA isolate. 2.4.2.1. Tissue digestion In general, DNA isolation employs a buffer containing one or more detergents, for instance SDS, NP-40 or Triton X-100. These detergents are used to break down the cell wall lipoprotein. Its anion-binding property permits detergent to precipitate some negatively charged compounds such as proteins and polysaccharides. TNES is the most used buffer for cell digestion. Several studies used 500-600 l of TNES consisting of 10200 mM Tris HCl pH 7.5-8.0, 125mM NaCl, 10-100mM EDTA, and 0.5-1% SDS to extract 50-100 mg of fish tissue, fin and scale (DeSalle et al., 1993; Asahida et al., 1996; Hsieh et al., 2003). SDS as detergent breaks apart fatty membranes of the cells while NaCl (source of salt and metal ion) is intended to increase the osmotic pressure outside the cell and help break apart the membranes. Concentrated salt solution changes the polarity of the solution where DNA dissolves in, whereas fats, carbohydrates and proteins do not. Since DNase is dependent on Mg2+ and Ca2+, the presence of EDTA (at least 2mM) in lysis buffer is critical to chelate these cations and thereby prevents the degradation of high molecular weight DNA (Merente et al., 1998). In addition, buffers maintain the solution in alcaline pH to retain DNA in aqueous phase. The presence of SDS, EDTA, metal ions and certain pH range is critical for the activity of enzymes involved in the DNA extraction (Deshpande et al., 2001). Occasionally, 4-8 M urea is involved to breakdown hard tissues such as fins and scales (Asahida et al., 1996; Hsieh et al., 2003). Alternatively, guanidinium thiocyanate is often used for DNA extraction as it has strong chaotropic properties (Botwell, 1987). Introduced by Chomczynki and Sacchi (1987), it is used as protein denaturant, acting as RNase inhibitor, and protecting cellular transcripts from degradation during tissue extraction. Nevertheless, since guanidinium
thiocyanate is categorized as harmful chemical, working with this method needs extra protective equipment, such as goggles, fume hood, and proper gloves.
CTAB (cetyltrimethyl ammonium bromide) is frequently applied as surfactant in DNA extraction. It is used for tissue digestion, together with -mercaptoethanol and polyvinylpyrolidone (PVP), followed by phenol-chloroform extraction. Originally, this method is developed for extraction of polysaccharide-contaminated samples, essentially plant tissue (Doyle & Doyle, 1987; Tel-Zur et al., 1999). PVP or activated charcoal or a
13
combination of both is often used in order to remove polyphenolics from further extraction steps. This method is, besides laborious, limited by the use of hazardous reagents. Konat et al. (1990) described a method that embeds the nuclei in agarose in order to protect the DNA from mechanical shearing during isolation. In this procedure, Proteinase K in the presence of SDS is used to remove proteins and lipid from the embedded DNA. The limitation of this method is that it is only suitable for suspension cells, neither for adherent cells nor tissues. The requirement of numerous buffer changes also makes this method laborious and time consuming (Merente et al., 1998). 2.4.2.2. Separating proteins and contaminants Proteinase K and RNase are two important enzymes involved in DNA extraction. Proteinase K is essential to remove protein from the embedded DNA while RNase is important to disrupt RNA that could interfere in the downstream applications. Proteinase K is an endolytic protease that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic or hydrophobic amino acids. Proteinase K is active in the presence of metal ions, SDS, urea, chelating agents (e.g. EDTA), sulfhydryl reagents and trypsin or chymotrypsin inhibitors. In spite of stable over a wide pH range (4-12.5), Proteinase K autolysis occurs increasingly at alkaline pH. The optimum temperature for proteinase K is 55-65C (Merante et al., 1998). In contrast, most other enzymes (e.g. DNase, RNase) are denatured under this condition. Therefore, the combination of Proteinase K and detergent buffer in elevated temperature are essential for the inactivation of endogenous nuclease. Commercially, proteinase K is produced by fungus Tritirachium album (Ebeling et al., 1974). Ribonuclease (RNase) is an endonuclease that cleaves the 3 end of cytosine (C) and uracil (U) residues. It is commonly used in molecular biology applications such as the removal of contaminating RNA from DNA preparations (Lee et al., 1996). RNases are ubiquitous in the environment as they are present in many biological materials. For example, the pancreas is rich in RNase (>1 mg/g tissue) and is the source for most commercially produced RNase. A study reported that RNase increases its activity in the presence of approximately 2 M urea (Deshpande et al., 2001). Since the optimum temperature of RNase is 37-45C, it must be applied separately from Proteinase K. Likewise, Proteinase K rapidly inactivates nucleases including RNase. Once cellular
14
proteins and lipids are separated, contaminating RNA can be degraded by DNase-free RNase followed by a chloroform extraction to remove the RNase. Studies revealed that concentration of Proteinase K used for DNA extraction varies depending on the kind of sample, time and temperature of incubation, and the presence of RNase. A concentration of 50ng proteinase K per l lysis solution was used to extract 50100mg of total DNA from fish tissue and fish fin with no addition of RNase, carried out at 37-42C incubation for overnight (8-16 hours) (Asahida et al., 1996). Wasko et al. (2003) examined 75ng/l proteinase for 100-300mg of fish fin and scales at 42C for 10 hours followed by the same concentration of RNase (42C for 1 hour) while Hsieh et al. (2003) worked with 400ng/l on fish tissue without RNase. In addition, Imai et al. (2004) employed relatively high concentration of Proteinase (4000ng/l) for digesting crab (genus Scyla) samples at 55C for 3 hours followed by 1000ng/l RNase at 37C for 30 minutes. 2.4.2.3. Precipitation and recovery of DNA In many classical DNA extraction procedures, organic solvents, mainly phenol and chloroform, are used to remove contaminants. For this purpose, phenol must be used in alkalic conditions (pH 7.5) otherwise the isolated DNA will remain in organic phase and interface (Blin & Stafford, 1976). In practice, a phenol: chloroform mixture, commonly added by isoamyl alcohol (P:C:I=25:24:1), at equal volume as lysis buffer are added to DNA extract to form a biphasic mixture (Asahida et al., 1996; Hsieh et al., 2003; Wasko et al., 2003). Isoamyl alcohol is important to prevent the mixture from foaming. Through a centrifugation at high speed (>10.000 rpm for 5-10 minutes), proteins and lipids will separate into the organic phase while the DNA, and other contaminants such as salts and sugars, remain in the aqueous phase. This is sometimes repeated depending on the requirements of the downstream processes, and then followed by extraction with chloroform: isoamyl alcohol (C:I=24:1) to remove the residual phenol. The aqueous phase containing DNA is concentrated by double volume of absolute ethanol to precipitate the DNA. The polarity of DNA renders insoluble in ethanol which is relatively less polar. The DNA precipitation is due to the interaction between ethanol and water resulting in less capability to dissolve the DNA. Isopropanol can be used instead of ethanol. In spite of the higher precipitation efficiency than ethanol, isopropanol is less volatile and therefore needs more time to dry the isolated DNA. By centrifugation, the DNA is concentrated and forms a white or transparent pellet. The next step is purification
15
using 70%-80% ethanol to remove salts present in the pellet. After air dried, purified DNA is re-suspended in TE (Tris-HCl and EDTA) buffer. Phenol-chloroform based extraction is proven to produce high yield of DNA with relatively good quality for PCR and downstream applications (Asahida et al., 1996; Wasko et al 2003). However, beside relatively laborious due to numerous changes of phenol and chloroform, this precipitation method has drawback as phenol and chloroform are hazardous and inconvenient materials. Thereby, extra caution is critical in order to handle the problems. 2.4.2.4. Commercial kits Many companies have developed commercial kits for total DNA extraction and purification. The protocols that are available commercially for example Wizard DNA Extraction kit and Wizard Magnetic DNA Purification for Food (Promega; Milan, Italy), DNeasy Tissue kit and QIAamp DNA mini kit (Qiagen; Hilden, Germany), Chelex (BioRad; Marne la Coquette, France), ABI PRISM 6100 Nucleic Acid Prep Station (Applied Biosystems Inc.), and genomic DNA purification kit (BioNobile; Turku, Finland). Based on the type, commercial kits can be classified in three categories i.e. manual commercial kit, manual automated, and fully automated (Mattocks, 2007). Factors governing the choice of kit are the speed of extraction, cost, reliability, sample requirement, sample tracking, storage requirement, and the quality of isolated DNA. Most of manual commercial kits work based on liquid-liquid phase extraction. The difference with classical procedures is the use of ready-to-use reagents (lysis buffer, precipitation reagents/washing buffer, and elution buffer) which minimize the work and time of preparation. Moreover, commercial kits allow convenient work as they commonly offer simpler procedures to provide purer DNA with safer reagents, importantly in avoiding the use of phenol-chloroform. In comparison to manual commercial kits, manually automated one has the advantages of the use of liquid-solid phase, instead of liquid-liquid phase extraction. Silica based column and magnetic beads are two important solid phases used to embed and bind the DNA. Column based methods generally rely on the use of lysis solution, binding buffer, washing buffer and elution buffer. After digestion with lysis solution, guanidinium thiocyanate is commonly used as chaotropic salt to bind the DNA. Bound DNA is purified by washing with buffer containing ethanol (Bio-Nobile, 2007). A special silica-glass
16
column then binds the DNA prior to its elution using a low salt buffer. This protocol eliminates the need of organic solvent extraction, DNA precipitation, and minimizes centrifugation (Roche, 2003). The DNA-bead interaction is based upon the specific affinity of the ligand on the surface of the beads. The beads can be coated with binding elements such as primary or secondary biotinylated antibody, streptavidin and protein (A or G). Streptavidin beads attach via a DNA linker simplifying bead removal by cleavage with DNase (BioNobile, 2007). Streptavidin magnetic beads present in different sizes (50 nm-3 m in diameter depending on the type of sample: cells, nucleic acids, or proteins) allow to minimize nonspecific binding of nucleic acid or protein to help prevent bead aggregation. Since the separation of the beads from the aqueous phase occurs with a magnet, centrifugation is not required. These kits may obtain DNA in less than one hour. It has been shown that DNA analysis by a fully automated extraction, BioRobot EZ1 DNA Tissue kit (Qiagen Corporation, Valencia, CA) and MultiProbe II Plus EX (Perkin Elmer, USA) for instance, proves time saving and reduces the risk of possible contamination (Pizzamiglio et al., 2006). The automation of the process allows minimizing manual interactions by means of robotic liquid handling, integrated shaking, heating, and vacuum filtration. 2.4.3. Determination of DNA yield and purity Several methods have been established for determination of DNA quantity and quality, such as spectrophotometric methods, radioactive labeling, and fluorimetric methods. Traditionally, spectrophotometry (UV-Vis) and spectrofluorometry are techniques commonly used for quantifying DNA due to their simplicity and less cost. Spectrophotometry technique measures DNA based on the determination of absorbance at 260 nm, the maximum absorbance of nitrogenous bases under UV light (Figure 2-4). On the other hand, spectrofluorometry relies on the fluorescent signal created by fluorescent dye-dsDNA binding at 480nm (excitation) and 520nm (emission). A number of reagents are used as fluorescent dye including ethidium bromide (EtBr), cyanine, Hoechst-33258, protamine, and hypocrellin A (Zhu et al., 1997). Both spectrophotometry and spectrofluorometry have advantages and disadvantages. It has been shown that spectrophotometry is the cheapest way to quantify DNA concentration and also simple as it can be acquired without the use of reference/DNA
17
standard. Since the concentration of pure dsDNA is 50g/ml (when A260=1.0), concentration of unknown DNA can be formulated as follows (Merante et al., 1998): DNA (g/ml) = A260 x l (light path in cm) x dilution factor x 50 The major disadvantages of the spectrophotometric method are the contribution of nucleotides and single-stranded DNA to the signal, and the inability to distinguish between DNA and RNA. Conversely, fluorometric method has higher sensitivity since it only measures dsDNA embedded by fluorescent dye. Generally, the detection limit by classical spectrophotometer is around 5 g/ml dsDNA aliquot, while by spectrofluorometer it is 10ng/ml (Paul & Myers, 1982; Zhu et al., 1997). Certain dyes even demonstrate the detection limit as low as 25pg/ml (Invitrogen, 2007). Fluorometric method is suitable for selective targets, dsDNA, ssDNA or RNA, with less interference. Nevertheless, the use of this method is restricted by the relatively high cost of fluorescent reagents. A UV-vis spectrophotometer allows measuring DNA quality/purity based on the ratio of absorbance at 260 and 280nm (A260/280). Theoretically, contaminating protein absorbs UV light at +280nm whereas phenol at +270nm. Contaminated DNA with any of these molecules expresses increasing absorbance at 280nm. The A260/280 ratio between 1.8 and 2.0 suggests minimal contamination (Smith, 1998). Lower values indicate protein contamination, higher values RNA contamination.
1.5 1.2 260 nm 0.9 OD 0.6 0.3 ssDNA
RNA
DNA
dsDNA
Ex:480nm
200
225
250 275 Wavelength (nm)
300
320
500
600 Wavelength (nm)
700
Figure 2-4. Characteristic spectrum of DNA obtained by spectrophotometer (left) and spectrofluorometer (right) Gel electrophoresis can also be used to evaluate DNA purity as well as to estimate DNA concentration. By loading onto a 0.8-1.5% agarose gel, DNA fragments can be separated by electrophoretic charge based on their lengths (molecular weight). The intensity of the bands correlates to the DNA concentration. More recently, there has been
18
increasing interest in the use of alternative techniques of DNA quantification such as quantitative PCR, hybridization, pulse-field gel electrophoresis (PFGE), and threshold assay. These techniques are proven to be useful due to a lower detection limit and their robustness (Mehta & Keer, 2007). 2.4.4. Polymerase chain reaction The study of product authenticity based on genomic profiling has been rapidly developed and improved with the progress of molecular biological tools, in particular polymerase chain reaction (PCR). Invented in 1983 by Kary Mullis at the Cetus Corporation, it offers a simple process to massively enrich a targeted fragment of DNA (Watson & Berry, 2003). PCR is an in vitro DNA amplification that involves a repeated cycling process of defined stages, termed denaturation, annealing, and extension. The reagents required for the PCR include DNA polymerase, each of four nucleotides (dNTPs), a primer couple, magnesium source, buffer solution and DNA template (Rapley, 1998). Rationally, when DNA is heated in excess of 94-95C for at least 60 seconds, the double strands come apart to produce two single strands (Rapley, 1998; Watson & Berry, 2003). This process is called denaturation, which allows the primers to bind to the DNA template (annealing), whose sequences are complementary to the primers. In the next stage the temperature is reduced to 35-60C for 30-120 seconds. Subsequently, the polymerase makes a complementary copy of the template DNA started from each primer, thereby creating a double strand of the target region. Known as extension, this step usually takes place at 72C for 60-180 seconds. In the next cycle, the dsDNA produced from the previous cycle becomes new template to produce a double new dsDNA (Figure 2-5).
1st Cycle
2nd Cycle, etc...
primer primer primer
DNA template
Separate 2 DNA strands, add primer
Add DNA polymerase
Separate 2 DNA strands, add primer
Add DNA polymerase
Figure 2-5. The principle of PCR amplification
19
Recently, real time PCR (RT-PCR) has been widely applied for DNA amplification due to its robustness, simplicity, and sensitivity (Weder et al., 2001; Hsieh et al., 2007). RT-PCR allows both qualitative and quantitative analysis. This technique incorporates the amplification and fluorescent detection by a single instrument in a single tube with data recorded online. Hence, the use of gel electrophoresis to assess the targeted product can be eliminated. Real-time and traditional PCR methods have been developed into a key technology for identification of foods, including fish and seafood products. Specific DNA sequences of fish and seafood have been generated through PCR amplification by designing sitespecific primers, such as tuna/family Scombroidae (Bartlett & Davidson, 1991; Rehbein et al., 1997; Richardson et al., 2006), caviar (DeSalle & Birstein, 1996; Rehbein et al., 1997), salmonoid (Rehbein et al., 1997), flatfish (Cspedes et al., 1998), mollusc and arthropods (Meritt et al., 1998), puffer fish (Cheng et al., 2003; Hsieh et al., 2003), gadoid/family Gadidae (Aranishi et al., 2005), billfish/family Istiophoridae (Hsieh et al., 2005; Richardson et al., 2006) and teleost fish (Sevilla et al., 2007). 2.4.4.1. Primer design Since annealing is started from the primer, it becomes a critical part in PCR amplification. Primers are designed to recognize the targeted region. The primers used in the PCR are designed based on existing sequences of close similarities or evolutionary conserved sequences. Many online sequences can be obtained from genetic databases, such as GenBank, EMBL, BOLD, MitoFish, and CBOLs. Specifically, sequences of fish and seafood are available via FISH-BOL and FishTrace. Amino acid sequence information may also be used to design PCR primers (Rapley, 1998). Besides from GenBank, amino acid sequences can be provided from Mascot, PeptideMass, ExPASy, and so on. There are some considerations in designing primers. Generally, the primers should have a matched GC content of 40-60% and must not have the potential to form primerdimers or hairpin beacons (Rapley, 1998). Table 2-2 describes a number of primers used for the amplification of the cyt b region in fish and seafood products. Typically, 15-30 base primers anneal efficiently if the PCR enzyme is Taq Polymerase, however this number may change for enzymes with greater heat stability (Roche, 2006). Shorter primers (less than 15 bases) anneal very effectively but they may not be specific enough. In contrast, longer primers recognize targets specifically but tend to anneal with lower efficiency. Priming efficiency can be increased by a G or C, or CG or GC at the 3' ends. Design of 3
20
or more C or G at the (3') ends should be avoided since it may promote mispriming at G or C-rich sequences due to stability of annealing (Qiagen, 2005). Primer pairs are designed with matched melting temperature (Tm). The difference of 5oC or more leads to reduce the amplification (Rychlik et al., 1990). Tm and optimum annealing temperature (Ta,Opt) can be estimated as follows: Tm (C) = 2 x (number of [A+T]) + 4 x (number of [G+C]) Ta,Opt (C) = 0.3 x(Tm of primer) + 0.7 x(Tm of product) 25 Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood
Name of primer L14841 H15149 Cyt BL Cyt BH Cyt BL1 Cyt BH FB349 FB496 59-3 59-4 CytB1 CytB2
UCYTB144F UCYTB272R UCYTB151F UCYTB270R
Sequence (5-3)
AAAAAGCTTCCATCCAACCAACATCTCAGCATGATGAAA AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA
Targeted Size Fragment (mers) (bp) 31 30 26 25 26 25 23 26 34 30 26 25 20 20 23 23 20 20 21 21 28 26 23 29 23 28 23 23 26 376 358 358 148 123 358
Target species and Source of reference Vertebrate species (Kocher et al., 1989) Tuna/family Scombroidae (Bartlett & Davidson, 1991)
Salmon, mackerel, hering, cod (Bartlett & Davidson, 1992)
CCATCCAACATCTCAGCATGATGAAA CCCCTCAGAATGATATTTGTCCTCA CCATCCAACATCTCAGCATGATGAAA CCCCTCAAAATGATATTTGTCCTCA GTCGAATGAATCTGAGGAGGCTT CCRATTGGGTTGTTTGACCCTGTTTC

AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA GCTGGTACCTCTACAAAGAAACATGAAACA
Tuna, bonito, mackerel (Rehbein et al., 1995)

Cod, herring, sardine, tuna, bonito (Rehbein et al., 1997)
CCATCCAACATCTCAGCATGATGAAA CCCCTCAGAATGATATTTGTCCTCA TGAGSNCARATGTCNTWYTG GCRAANAGRAARTACCAYTC TGTGGRGCNACYGTWATYACTAA AANAGGAARTAYCAYTCNGGYTG GGAAAACCCATCCAATCCTA CAGCAACAACAAAGGGGAAT GCTATRCACTAYACMTCRGAC GCCTCCTCARATTCATTGGAC CCCTCTAATATCTCQGTCTGATGAAACB GCGTAGGCAAATAGGAARTATCAYTC TTCTCAGTAGACAACGCCACCCT CTAACCCGATTCTTTGCCTTCCACTTCCT CGATTCTTCGCATTCCACTTCCT GGTCTTTGTAGGAGAAGTATGGGTGGAA GGGGTAAAGTTGTCTGGGTCTCC GCGGGGGTAAAGTTGTCTGGGTC AGGAAGTATCATTCGGGCTTAATATG
Flatfish (Cspedes et al., 1998) Mollusc and arthropods (Meritt et al., 1998) Gadoid/family Gadidae (Aranishi et al., 2005)
Billfish/family Istiophoridae
430
TR-14F TR-571R L-CYTBF H-CYTBF CBF-A CB3-3 CytBI-6F CytBI-7F CytBI-1F CytBI-5R CytBI-3R CytBI-2R CytBI-4R
558 348 715
(Hsieh et al., 2005) Scombroidae, Istiophoridae (Richardson et al., 2006)
700-750
Teleost fish (Sevilla et al., 2007)
Wobbles: Y: T/C; M: A/C; R: G/A; K: T/G; W: A/T; S: C/G; H: A/C/T; V: A/C/G; D: A/G/T; B: C/G/T; N: A/C/G/T
21
Not only for specific amplification, but primers are also important for post-PCR application, such as direct sequencing and fingerprinting analysis. In some cases, a degenerate primer is useful to overcome the limited sequence information of such species/families (Knoth et al., 1988). By introducing alternative bases (wobbles) as inosine at a particular position, degenerate primers are advantageous for amplification of wide range of families (Crick, 1966). A number of computer software are currently available to aid in primer design, for instance DNAsoftware, PrimerDesign, EMBL, PremierBiosoft, and OligoDesign. 2.4.4.2. Components of PCR reaction During PCR amplification an enzyme is used to carry out the extension of the fragment. Historically, DNA polymerase-I was used for this purpose. However, because it is heat labile, fresh enzyme was required during each cycle which made the technique laborious and costly (Rapley, 1998). The introduction of thermostable DNA polymerase transformed PCR technique to allow full automation. The first introduced thermostable DNA polymerase was isolated from the bacterium Thermophylus aquaticus, namely Taq DNA polymerase which has optimum temperature of 72C (Eckert & Kunkel, 1990). The higher thermal stability, the more useful is the enzyme, especially for amplifying GC-rich regions where a high denaturation temperature is required. Several thermostable DNA polymerases are produced from the different sources, such as Thermococcus litoralis, Thermotoga maritime, and Thermus thermophilus (Rapley, 1998). Magnesium (Mg2+) is another important element in the PCR amplification. It is not only required to form a complex with the dNTPs which is critical for incorporation in the extension step of the PCR cycle but it also affects the specificity of the primer-template interaction and the denaturation of the dsDNA (Rapley, 1998). Insufficient Mg2+ results in low yields while an excess of it creates nonspecific products. Generally, a concentration of 1-4 mM MgCl2 is used, depending on the DNA template and the primers. The pH of the PCR reaction is an essential factor since changes in pH will affect the amplification efficiency, in particular of long fragments (Cheng et al., 1994). In order to maintain the pH at 8.3, at room temperature, a buffer/salt containing 50 mM KCl and 10 mM Tris-HCl is used. The PCR efficiency may also be reduced by contaminants present in the template or caused by improper handling.
22
Essentially, application of RT-PCR requires fluorescent dye to detect amplified product. There are two classes of fluorescent assays in RT-PCR, known as sequenceindependent and sequence-specific probe. Sequence-independent assays rely on fluorophores, SYBR Green I for instance, that binds to entire dsDNA molecule instead of oligonucleotide. Conversely, sequence-specific assay rely on oligonucleotide probes that hybridize to their complementary sequence in the targeted products. Fluorescent Resonance Energy Transfer (FRET) and HyProbe are examples of sequence-specific probes (Roche, 2006). 2.4.5. PCR product analysis Traditional PCR requires post-run visualization to assess the targeted fragment (amplified product). Generally, DNA strands are able to be separated and visualized by the application of polyacrylamide-GE, agarose-GE, or pulse field-GE (Smith, 1998). Confirmation of the targeted PCR product is normally performed by using GE. AgaroseGE separates a wide range of DNA fragments (0.1-40 kb), which is preferable to polyacrylamide-GE (5-400bp). An agarose concentration of 0.3-3% (w/v) in TAE buffer 1x is commonly used to separate DNA fragments. The longer the fragment the lower the concentration of agarose required. A set of apparatus is needed to carry out the electrophoresis. Recently, the submarine gel system is universally used for agarose-GE (Smith, 1998). In this system, the prepared agarose gel on a supporting plate is submerged into a chamber containing electrophoresis buffer (TAE 1x). The wells are created in the agarose gel with the aid of a comb inserted into the cooling agarose. Into these wells, samples which have been mixed with a loading dye are loaded. Electrophoresis is carried out for certain period, varying considerably from 30 minutes to hours, depending on the size of the gel, gel concentration, and electrical charge. The DNA fragment migrates from the negative charge to the positive charge. An intercalating dye is included in the electrophoresis to visualize DNA fragments. The dye intercalates the stacked bases of dsDNA to produce fluorescence when illuminated under UV light (254-300 nm). A particular concentration of dye can be mixed within the gel, running buffer or combination of both. EtBr (3,8-diamino-6-ethyl-5-phenylphenanthridium bromide) was commonly used for DNA staining. However, EtBr is known as powerful mutagenic and potential carcinogen. Recently, lower mutagenic dyes such as
23
SybrSafe, SybrGreen, SybrGold (Molecular Probes) and GelRed, GelGreen (Biotium Inc.) are established commercially, despite their costs are higher than EtBr. A successful PCR amplification results a strong band of targeted amplified product with faint bands of residual nucleotides and primers. Side bands or non amplifiable band indicates that amplification is not optimal. Overload of DNA will result in smear-bands while excessive salt will lead to necking bands (Smith, 1998). Since GE only assesses targeted fragment, it does not give species/product specific information. Further analysis is required to assess the amplified products, particularly based on the nucleotide profiles. Direct sequencing and fingerprinting techniques are commonly used for analyzing PCR product. 2.4.5.1. DNA sequencing DNA sequencing is the most informative and precise technique for species identification. DNA sequencing is a modification of DNA replication, which differs from normal replication in the inclusion of dideoxynucleotides (ddNTPs) instead of dNTPs (Sanger et al., 1977). The different between ddNTP and dNTP is the lack of a 3-OH group. When ddNTP is incorporated into DNA, the synthesis is terminated, the process known as termination sequencing. Sequencing is carried out in four reactions, each of which contains all four dNTPs but only one of ddNTP. Thus, the reaction products are four nested sets of fragments which are terminated according to the ddNTP included in the reaction. Historically, sequencing involves cloning the DNA into a suitable vector to create DNA in a format that can be used for sequencing. There are two categories of cloning vectors, known as single-stranded and double-stranded vectors. Plasmid of a bacterium, such as E. coli, is the most used double-stranded vector for DNA cloning. On the other hand, a single-stranded vector can be obtained from viruses or bacteriophages (Rapley, 1998). Recently, PCR products can be used as a sequence template. This technique, called direct sequencing, eliminates the cloning stage and thereby significantly saves time and materials. The disadvantages of direct sequencing are the limited fragment size (10002000bp) and the need of PCR products purification. Purity and sufficient concentration of amplified product becomes the most critical factor for the efficiency and reliability of the sequencing method (Rapley, 1998).
24
In order to visualize the sequence, a detectable label is incorporated into the sequenced strands. There are three types of labels used to visualize the sequence: radioactive, four-color fluorescent, and one-color fluorescent. Radioactive based labeling uses isotope of
35 32 33
S,
P or
P to be incorporated to the ddNTP. The extended DNA is
visualized after electrophoresis by drying the gel and subsequently exposed it into a film sensitive to radioactivity. 35S is the most used isotope as it exposes low energy -emission with reasonable half-life (87.4d) (Spencer, 1998). Recently, the trend of sequencing is moving toward automated-sequencing system based on fluorescennce. Fluorescent sequencing relies on excitation of fluorescent-labeled DNA strands when exposed to laser light. The major advantage of this technique is that the sequence information can be collected directly while the gel is running (Spencer, 1998). Applied Biosystems/ABI (Perkin Elmer Corp), Amersham Pharmacia Biotech, and Li-Cor Biosciences are among recognized manufacturers of automated sequencer. For species differentiation, the sequence of DNA template is compared to references/libraries. The index of similarity between two or more sequences is used to analyze the most likely identification by using BLAST (Basic Local Alignment Search Tool). Sequencing enables species identification without reference material if the generated sequence is available in the database. The database can be created manually or obtained online. The most recognized online DNA databases are GenBank and EMBL, while Fishtrace specifically constructs sequence databases of commercially important fish based on cyt b and nuclear rhodopsin. Nonetheless, not all sequences of the species are available in several online DNA databases. Direct sequencing has been successfully used to identify tuna species (Bartlett & Davidson, 1991), adulterated dried mullet (Hsieh et al., 2003), specimens of tuna and billfish larvae (Richardson et al., 2006) and teleost specimen (Sevilla et al., 2007) based on the cyt b gene. Sequencing was also employed to identify Australian fish species for DNA barcoding purposes based on COI region (Ward et al., 2005). 2.4.5.2. Fingerprinting techniques The main drawback for large-scale implementation of sequence-based identification is cost requirement. Alternatively, several methods have been developed for species identification. These techniques, known as fingerprinting, include Denaturing/
Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single Strand Conformation
25
Polymorphism (SSCP), Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP), and Random Amplified Polymorphic DNA (RAPD) (Rapley, 1998; Spencer, 1998; Brooker, 1999; Mackie et al., 1999; Hird et al., 2005). By using these methods, the phylogenic information can be obtained more economically and rapidly (Hwang & Kim, 1999). DGGE employs the use of a polyacrylamide gel with a gradient of denaturant to separate the targeted DNA based on its stability against the denaturant. DNA fragments rich in GC will be more stable and remain double-stranded until reaching higher denaturant concentrations (Rapley, 1998). A mixture of ureum-formamide is used as denaturants in the DGGE technique. Denatured DNA molecules become effectively larger and slow down or stop in the gel and thereafter DNA fragments of different sequences can be separated. The system is very sensitive to detect a single mutation in several hundreds of base pairs of DNA (Smith, 1998). Similarly, TGGE is generated based on the gradient temperature (Rapley, 1998). PCR-RFLP is the most widely used method for identifying different fish (Hsieh et al., 2003). This technique relies on the use of restriction enzymes which cleave dsDNA at defined base pairs to generate species specific DNA fragments. Differences of base pair between species can be detected by choosing a restriction enzyme which cuts the DNA at particular sites. Endonucleases such as AluI, EcoRI, HaeIII, HindIII, SmaI, etc, are commonly used for restriction analysis of amplified PCR products (Rapley, 1998). The EcoRI, an enzyme produced by E. coli RY13 for example, recognizes every site consisting GAATTC. After digestion for several hours at 37C, the resulting fragments are separated by electrophoresis in an agarose gel containing fluorescent dye. The advantage of this method is that it is suitable for some functional genes which are not resolved by DGGE analyses (Brooker, 1999). RFLP may also overcome problems with degenerate primers that can plague some DGGE methods. PCR-RFLP enables to identify a single point mutation since a restriction site would change the restriction pattern and a clear identification becomes possible (Brooker, 1999). PCR-AFLP/RFLP analysis of the cyt b gene has been used for identification of fish species (Ram et al., 1996; Cespedes et al., 1998; Hsieh et al., 2003). In SSCP, dsDNA is denatured by denaturing solution containing formamide, bromphenol blue, and xylene cyanol in NaOH to form ssDNA. The ssDNA is then folded such that complementary sections are bound together. Differences in the three dimensional
26
structure caused by alterations in base pair sequences of the single strand lead to differ the electrophoretic mobility, which are visualized by silver staining. In comparison to other fingerprinting techniques, SSCP is relatively cheap and fast to perform. Differentiation of canned tuna and bonito has been achieved by this method (Rehbein et al., 1995). In addition, the RAPD technique uses a short and not specific primer to generate unique band patterns of amplified DNA (Mamuris et al., 1999). However, the use of SSCP and RAPD techniques is limited by the necessity of reference material (Rehbein et al., 1995). 2.4.5.3. Other techniques There are relatively new technologies which work in the similar way as finger printing. Denaturing HPLC (PCR-DHPLC) employs an HPLC column rather than acrylamide gel for separating PCR products (Barlaan et al., 2005. Separation of amplified products is according to the elution of partially melted DNA bases. Since typically GC-rich DNA denaturizes at higher temperature, it leads to the reduction of dsDNA and therefore the elution of the sample will be faster. This technique was originally developed for mutation analysis (Frueh & Noyer, 2003). Not only it is able to generate targeted DNA fragment, RT-PCR also offers some advantages in comparison to classical PCR. Online information, chiefly the amplification curve and melting points, can be provided while PCR is running. Since melting point (Tm) is basically sequence dependent, it permits to discriminate between different PCR fragments. RT-PCR has been satisfactorily used to identify fish fillet from grouper, tuna fishes, and commonly substitute species (Lopez & Pardo, 2005; Trotta et al., 2005).
27
CHAPTER III OBJECTIVES OF PRESENT STUDY

3.1. Problems Primer design plays an important role in the PCR amplification and contributes significantly to the downstream applications of product identification. The previous literature reviewed various primers have been developed specifically to amplify specific groups of fish and seafood products. However, several fish identities are not resolved by existing primers, for instance Microstomus kitt, Scopthalmus maximus, Hyperoplus lanceolatus and Ammodytes tobianus (Hoffman, personal communication). In addition, studies on the genomic identification of elasmobranches, crustaceans and molluscs based on cyt b gene are very limited. The studies on the identification of crustacean and molluscan taxa focusing on cyt b were described by Meritt et al. (1998) and Nguyen et al. (2002), while the studies on the identification of elasmobranches were focused on shark (Martin & Palumbi, 1993; Heist & Gold 1999; Briscoe et al., 2005). With regard to the fact that there is no universal primer couple available for the majority of fish, crustaceans and molluscs at present time, it is essential to provide and validate it. Universal primers are valuable for identification of samples that are unrecognizable by morphological characters such as filleted products, surimi and other processed/mixed products. It is also advantageous for identification of a wider range of species or species that are not resolved by existing primers. 3.2. Objectives The main goal of this study is to develop standardized universal primers for species identification of fish and seafood (teleost fish, elasmobranches, crustaceans and molluscs) by polymerase chain reaction base on cyt b mitochondrial DNA. Furthermore, since DNA quality, primers suitability and amplification condition are among the crucial factors in the success of PCR amplification, this research aims to: a. evaluate the different DNA extraction methods, b. optimize the existing primers for cyt b gene amplification suitable for fish, mollusc and crustaceans. Adapted primers were designed based on the evaluation of existing primers, and c. provide the optimal PCR protocol of the validated primers.
28
3.3. Research framework
Sample collection
Unknown biological references Sequence database - Evaluation of existing primers - Designing the degenerate primers - Validation of universal primers
Morphological identification (if available) Comparison of DNA extraction methods
DNA isolation
PCR/RT-PCR amplification
DNA quantity and purity
PCR product analysis
Sequencing
RT-PCR analysis
- Evaluation of targeted fragments - Index of similarity (BLAST)
- Evaluation of melting points (Tm) - Identification of dissimilarity
: Focus of the study
Figure 3-1. Framework of present study
29
CHAPTER IV MATERIALS AND METHODS

4.1. Construction of reference database of cyt b gene Genomic data bases of cyt b of various fish and seafood were collected via NCBI and FishTrace (Figure 4-1). In NCBI, cyt-b database is categorized in Nucleotide group on menu Search. Species of to be searched fish/seafood must be entered, for example: Gadus morhua cytochrome b, then Go must be clicked. The available database (sequence) must be displayed in FASTA to perform the sequences as fasta format. The fasta format sequence is then copied to Notepad in order to be able to integrate it into bioinformatics applications. The format of entry files is >gb nr|species name|reference number|cytb|data origin (NCBI or FishTrace). Meanwhile, FishTraces data base can be searched by either scientific name or common name. The cyt-b sequence is classified in Genetics group. The detail sequence, furthermore, must also be copied as FASTA format as explained previously. The provided sequences were used in designing the primers and evaluating the sequence results.
Figure 4-1. Genomic database obtained from NCBI (left) and FishTrace (right) 4.2. Sample collection and preservation Selected samples (finfish, molluscs and crustaceans) were obtained from either the North Sea or selected fish markets in Belgium. Fresh samples were identified by means of morphological features. Gutted and eviscerated samples were filleted prior to extraction. Three to six specimens of 14 species of fish, 7 species of crustaceans, and 6 species of molluscs were used for this work. Specification of the samples is described as follows:
30
a. Fish: - Round fish: cod (Gadus morhua), faneca (Trisopterus luscus), tuna (Thunnus sp.), and grey gurnard (Eutrigla gurnardus). - Flat fish: common sole (Solea solea), lemon sole (Microstomus kitt), and tarbot (Scophthalmus maximus). - Elasmobranches: thornback ray (Raja clavata) and smooth skate (Malacoraja senta). - Sand lances (smelt): small sandeel (Ammodytes tobianus), great sandeel (Hyperoplus lanceolatus), and 3 unknown smelt fish (US1, US2, and US3). b. Crustacean: - Brown shrimp (Crangon crangon), giant tiger shrimp (Penaeus monodon), king shrimp (P. Latisulcatus), giant river shrimp (Macrobranchium rosenbergii), deep-sea shrimp (Solenocera sp), and 2 unknown shrimp (990 and 991). c. Mollusc: - Blue mussel (Mytilus edulis), green mussel (Perna canaliculus), illex squid (Illex argentinus), and 3 unknown mussels (T1, 120 and 2251) The reason for choosing these species was that some of them have previously been well studied, such as G. morhua, Thunnus sp, and S. solea (Bartlett & Davidson, 1991; Rehbein et al., 1997; Cspedes et al., 1998; Richardson et al., 2006). Other species are known difficult to be identified by existing primers, for instance M. kitt, S. maximus, A. tobianus, H. lanceolatus, C. crangon and M. edulis. 4.3. Comparison of DNA extraction methods A commercial kit (Wizard DNA Extraction kit-Promega) and 3 classical methods based on TNES-Urea Phenol-Chloroform were evaluated to isolate the total DNA of 4 selected samples (S. solea, T. luscus, C. crangon, and M. edulis). These classical methods have been widely used to extract DNA either from fish tissue, fin, scales, or food products (Asahida, et al., 1996, Wasko et al., 2003; Hsieh et al., 2005). The best method, resulting in the best DNA quantity and purity for PCR application was then used in the further application. The chemicals, reagents and solutions used for this work are described in Appendix II (see page 70).
31
4.3.1. DNA extraction method 1 (Promega) Isolation of DNA was performed based on the manufacturers instruction (Promega, 2002). 120 l of a 0.5M EDTA solution pH 8.0 was added to 600 l of Nuclei Lysis Solution (NLS) in a 1.5 ml Eppendorf tube. Approximately 100 mg of tissue was added into each tube followed by 17.5 l of 20 mg/ml Proteinase K. The tubes were then vortexed gently for 20 second and incubated overnight at 55C. Subsequently, 3 l of 4 mg/ml RNase were added to the nuclear lysate and mixed gently prior to incubation at 37C for 30 minutes. The DNA was extracted by adding 200 l of Protein Precipitation Solution (PPS). The tubes were then vortexed vigorously at high speed and followed by a centrifugation at 14.000 rpm for 10 minutes. Supernatant was transferred to a new tube containing 600 l of isopropanol (IPA). After being chilled at -20C for 1 hour, the DNA was precipitated at 14.000 rpm for 10 minutes. The DNA pellet was washed with 600 l of 70% ethanol, air dried and resuspended in 100 l TE buffer.
4.3.2. DNA extraction method 2 (Hsieh et al., 2005) In a 1.5 ml Eppendorf tube, approximately 100 mg of tissue was crushed with 600 l TNES buffer (1% SDS). 15 l of 20 mg/ml Proteinase K was added to each tube followed by overnight incubation at 55C. The DNA isolation was performed by 600 l of phenol:chloroform:isoamyl alcohol (25:24:1). Rotation at 14.000 rpm for 10 minutes separated the undissolved materials. The top layer was transferred to a new tube followed by another extraction with 600 l of phenol:chloroform (24:1). The DNA was precipitated in 3M NaOAc pH 5.3 and two volumes of absolute ethanol. After being cooled at -20C for 1 hour, the tubes were centrifuged at 14.000 rpm for 10 minutes. The DNA pellet was washed in 600 l of 70% ethanol, air dried and resuspended in 100 l of TE buffer.
4.3.3. DNA extraction method 3 (Wasko et al., 2003) Approximately 100 mg of fresh tissue was grounded in a 15 ml falcon tube with 4 ml of TNES-Urea buffer (0.5% SDS; 4 M urea). 15 l of 20mg/ml RNase was added to the tube prior to an hour of incubation at 42C. After incubation, 75 l of 4 mg/ml Proteinase K was added to each tube followed by incubation at 42C for 10 hours.
32
The DNA was extracted with 4 ml of phenol:chloroform:isoamyl alcohol (25:24:1). After being inverted for 30 seconds, the tubes were rotated at 14.000 rpm for 10 minutes. The top layer was transferred to a new tube. The DNA was precipitated in 1M NaCl and two volumes of absolute ethanol. Centrifugation at 14.000 rpm for 10 minutes was done to recover DNA. The DNA pellet was briefly washed in 70% ethanol, air dried and resuspended in TE buffer and incubated at 65C for 1 hour.
4.3.4. DNA extraction method 4 (Asahida et al., 1996) Approximately 100 mg of tissue was transferred into a 1.5 ml Eppendorf tube. 600 l of TNES-Urea buffer (6 M urea; 1% SDS) was used as the digestion buffer. 2l of 20 mg/ml Proteinase K was added to the tube for protein digestion and the mixture then incubated overnight at 37C. After this period, 3 l of RNase was added to the tubes to and the tubes were maintained at 37C for 30 minutes. The DNA was isolated by adding 600 l of phenol:chloroform:isoamyl alcohol (25:24:1) to the tube. After being inverted for 20 seconds, it was rotated at 14.000 rpm for 10 minutes. The top aqueous layer was transferred to a new tube followed by the second extraction by adding 600 l chloroform:isoamyl alcohol (24:1). After the centrifugation, the top layer was transferred again to a new tube. DNA precipitation was done by adding 1/10 volume of NaOAc 3M, pH 5.3 and 2 volume of 99% ethanol. After being gently inverted, the tube was stored at -20C for 1 hour prior to be rotated at 14.000 rpm for 10 minutes. The DNA pellet was washed in 70% ethanol followed by air drying. The DNA was resuspended in 100 l of TE buffer, incubated at 65C for 1 hour and ready for downstream applications.
4.3.5. Determination of DNA yield The DNA concentration was determined by spectrofluorometry. Relative fluorescence
was measured by using a
Shimadzu
Fluorospectrophotometer
(RF1501).
The
spectrofluorometric method was performed using Picogreen dye specifically for dsDNA (Invitrogen) and measured at 480nm excitation and 520nm emission.
33
4.3.5.1. Standard curve 2 g/ml of Lambda DNA (Invitrogen) was used as working solution to create a standard curve. A seven-point standard curve (0; 5; 10; 15; 20; 25 and 30 ng/l) was prepared by diluting the working solution in the same way as the experimental sample (Table 4-1). After incubation for 5 minutes at room temperature and protected from light, samples were measured. The fluorescence values were used to generate the standard curve (Figure 4-2) of relative fluorescence versus the DNA concentration of the samples as follows: y = ax + b, where y is DNA concentration while x is relative fluorescence (RF) Table 4-1. Protocol used to prepare the standard curve DNA Conc. (ng/ml) 0 (blank) 5 10 15 20 25 30 2 g/ml DNA Stock (L) 0 5 10 15 20 25 30 TE-buffer 1X (L) 1998 1993 1988 1983 1978 1973 1968 PicoGreen dsDNA Reagent (L) 2 2 2 2 2 2 2 Total Volume (L) 2000 2000 2000 2000 2000 2000 2000
700 600
Relative Fluoroscence
Y = 20.592X + 9.1132 R2 = 0.9993
500 400 300 200 Ex : 480nm Em : 520nm 1 00 0 0 5 1 0 1 5 20 25 30 35
DNA (ng/ml)
Figure 4-2. An example of standard curve for spectrofluoromric measurement
34
4.3.5.2. Sample Analysis One l of experimental DNA solution was diluted in 1997 l TE buffer 1X to a final volume of 1998 l in a disposable cuvette (ROTH; Art.8128). 2 l of PicoGreen dsDNA quantitation reagent was added to each sample and the samples were then incubated for 2 to 5 minutes at room temperature. The relative fluorescence was measured by using instrument parameters that correspond with those used for generating the standard curve. The DNA concentration of the sample was calculated from the DNA standard curve through the following formula: DNA (ng/l) = [(RF-b)/a] x 2; where RF is relative fluorescence
4.3.6. Evaluation of DNA purity The DNA purity was estimated by spectrophotometry. A NanoDrop ND-1000 spectrophotometer (Isogen) was used to calculate the ratio of absorbance at 260 and 280 nm. The analysis was carried out according to the manufacturers instruction (NanoDrop, 2007). 1 l sample was pipetted onto the lower pedestal/the receiving fiber (Figure 4-3). The integrated software measures the DNA concentration and the ratio of A260/280.
Sampling arm
Lower measurement pedestal upper measurement pedestal
(a)
(b)
(c)
(d)
Figure 4-3. The application of NanoDrop (a-c) and its typical data output (d) In order to evaluate DNA integrity, including degraded DNA and contaminants, the DNA isolates were checked by agarose GE. 1% agarose (Promega) was prepared in 1X TAE buffer with Gel Red staining dye (Molecular Probes). For a 20x10 cm2 gel, 1 g of agarose was diluted in 67 ml of TAE buffer 1.5X and 33 ml of GelRed in a 500 ml Erlenmeyer flask. The mixture was heated in a microwave for +5 minutes until the agarose was completely dissolved. After cooling down for a few minutes, the gel was poured into the gel mold with casting comb in place. The gel was allowed to cast for 15-20 minutes, placed in the chamber of Midicell Primo EC-330 (Thermo) (Figure 4-4). The reservoir was filled with 1X TAE buffer until the buffer just covered the gel. 5 l of samples were prepared and to each of them was added one drop of blue-orange loading dye (Promega). A
35
Smart Ladder 1800 (Eurogentec) was used as a 1 kb DNA marker. The gel was electrophoresed for 1 h at 66 V until the orange dye reached approximately 3/4 of the gel. DNA fragments were visualized on a Consort UV transilluminator at 254 nm and captured by Edas-290 digital camera (Kodak).
A. Thermal cycler
B. Electrophoresis chamber
C. UV illuminator
Figure 4-4. Apparatus for PCR applications
4.3.7. PCR assay A PCR assay was employed to evaluate the efficiency of DNA isolate to amplify the targeted fragment. The CytBL1/CytBH primer couple was used to generate ~357bp of the cyt b gene. The reaction was performed in a final volume of 20l. 10l of Jump Start RED Taq Ready Mix (Sigma P-0982) was used to obtain 10mM Tris-HCl pH 8.3, 50mM KCl, 2mM MgCl2, 0.2mM of each dNTP, and 0.03U/l Taq DNA polymerase. 2 l of each primer (10mM) and serial dilution (5 and 20 ng/20l) of DNA templates were added following the instruction as described in Table 4-2. Amplification was carried out in a PCRexpress thermal cycler (Hybaid). The PCR conditions included initiation (94oC for 5 min), 35 cycles of amplification (94oC for 30 sec; 50oC for 30 sec; 72oC for 1 min) and final extension (72oC for 5 min). Table 4-2. Composition of PCR reaction Reagent 2x JumpStart REDTaq ReadyMix Forward primer (10 mM) Reverse primer (10 mM) DNA template PCR H2O Total volume Volume (l) 10 l 2 l 2 l variable variable 20 l Final concentration 1x 1 M 1 M 5 and 20 ng -
36
4.3.8. Visualization of PCR product Amplified PCR products were analyzed by gel electrophoresis on 2% (w/v) agarose (Promega). The preparation of the gel and visualization of the result was as described in Section 4.3.6. (see page 35). 5 l aliquot of amplified products was loaded on the gel together with a 100 bp 1700 DNA ladder (Eurogentec) as the marker.
4.4. Optimization and validation of a universal PCR protocol 4.4.1. Primer design At first, the primer couple of CytBL1/CytBH was evaluated and optimized to amplify the selected samples. This primer couple has widely been used to amplify ~357bp of the cyt b of various species (Kocher et al., 1989; Bartlett & Davidson, 1991 & 1992; Cspedes et al., 1998; Richardson et al., 2006). Since this primer was not optimal for crustacean, mollusc, and some fishes, degenerate primers were designed by introducing some wobbles. The primers were designed by means of Oligo software following a number of rules for primer design (Qiagen, 2005; Roche, 2006). Alternatively, other universal primers (UCYTB) were also validated to amplify ~410bp of the cyt b gene. The primers were designed based on those described by Meritt et al. (1998). Table 4-3 describes the properties of the primers used in this study. In addition, the scheme of targeted fragments created by the validated primers is drawn in Figure 4-5.
37
(5) 2 &,6 ~ 357bp 1, 3, 4, 5 (3)

70
427
827/830/833
(3)
9 - 12 ~ 410bp 7&8
421/424
(5)
Figure 4-5. The scheme of targeted fragments generated by the evaluated primers 4.4.2. PCR assay The PCR reaction was performed in a final volume of 20 l as described in Section 4.3.7. (see page 36). The PCR conditions were adjusted depending on the set of primers as described in Table 4-4. Table 4-4. PCR condition of the different primer sets
Primer CytBL1 CytBH CytBL1A CytBL1B CytBL1C CytBHW UCYTB151BF UCYTB152BF UCYTB270B UCYTB271R UCYTB272BR UCYTB273R PCR condition Initiation Denaturation Annealing Extension Final extension Initiation Denaturation Annealing Extension Final extension 94oC; 5 minutes; 1 x 94oC; 30 seconds 45-60oC; 30 seconds 72oC; 1 minute 72oC; 5 minutes; 1 x 94oC; 4 minutes; 1 x 94oC; 1 minute 45-60oC; 1 minute 72oC; 2 minute 72oC; 5 minutes; 1 x
35 cycles
35 cycles
Amplified PCR products were analyzed by gel electrophoresis on 2% (w/v) agarose (Promega). The preparation of the gel and visualization of the result was as described in Section 4.3.6. (see page 35). 5 l aliquot of amplified products was loaded onto the gel together with a 100 bp 1700 DNA ladder (Eurogentec) as the marker. 4.4.3. Direct sequencing of PCR product Direct sequencing was attempted to confirm whether the designed/validated primers amplify the targeted fragments. Sequence analysis was carried out using a 4 color dye (Big Dye, Applied Biosystems) and performed in an ABI 3730xl DNA analyzer at AGOWA GmbH Berlin, Germany. Prior to the sequencing, PCR products were purified using the High Pure PCR Product Purification Kit (Roche Applied Sci. Mannheim, Germany) according to the manufacturers instructions.
38
Sequence chromatograms were viewed and evaluated by using ChromasPro and BioEdit software (Figure 4-6). Both forward and reverse sequences were edited by trimming out the ambiguous bases. The selected sequences were then assembled to analyze the overlapping bases.
Figure 4-6. Software for sequence analysis: ChromasPro (left) and BioEdit (right)
4.4.4. Real Time PCR application Prior to the RT-PCR amplification, all reagents (2x QuantiTect SYBR Green RTPCR Master Mix and primers) were thawn. The master mix was prepared to a final volume of 20 l according to the manufacturers instructions (Table 4-5). Into each microplate well, 15 ul of the mix solution were filled. 5 l of DNA template (final concentration of 10ng/20l) was added to each well, except the blank. The microplate was centrifuged for 3 min at 3000 rpm (Sigma 3-18K; Sartorius). The RT-PCR amplification was carried out in a LightCycler 480 (Roche) at the conditions as described in Table 4-6. The melting curve was determined from the thermal profile observed for 30 seconds after elongation (60-90C with ramp rate 2.2-4.4C/s). The melting points (Tm) generated from the melting curve (Figure 4-7) were used to discriminate between closely-related species. Table 4-5. Reaction components of RT-PCR amplification Component SYBR Green RT-PCR Master Mix 2x Forward and reverse primer 10M DNA template PCR grade H2O Final volume Volume/reaction 10l 1l 5l 3l 20 l Final concentration 1x 0.5M 10ng To make final volume 1 x Reaction Mix
39
Table 4-6. Thermal profile of RT-PCR amplification Step Initial activation Denaturation Annealing Elongation Cycle number Time 10 min 10 sec 10-20 sec 30 sec 30-40 cycles Temperature (oC) 95 95 45-60 72 Remark Ramp rate 4.4C/s Ramp rate 4.4C/s Depends on the melting temperature Ramp rate 2.2C/s Ramp rate 4.4C/s Depends on the length of the targeted fragment
Figure 4-7. Typical results of RT-PCR: annealing curve (left) and melting curve (right)
4.5. Data analysis The similarity index of selected samples based on sequence profile was compared to the references/libraries obtained via GenBank and FishTrace by using BLAST analysis (Figure 4-8).
Figure 4-8. An example of BLAST analysis obtained via GenBank (left) and FishTrace (right)
40
CHAPTER VII CONCLUSIONS

Despite yielding lower DNA concentration, the commercial kit produced better quality of DNA isolate compared to the classical methods, performed by higher efficiency in PCR amplification. In addition, commercial kits offer simpler procedure and avoid the use of harmful reagents in comparison to classical methods. In the classical methods evaluated in this study, 4 M urea in combination with 0.5% SDS (Wasko et al., 2003) or 1% SDS without urea (Hsieh et al., 2005) proved to be the optimal lysis solution for fish and seafood products. Typically, the CytBL1/CytBH primer couple worked effectively on most fish (except S. maximus, M. kitt, and sand lances/smelt fish). In contrast, these primers showed low efficiency in amplifying crustacean and molluscs. Adjustment of Mg2+ concentration did not affect significantly on annealing efficiency of C. crangon and M. edulis. Meanwhile, the degenerate primer couple CytBL1C/CytBHW effectively amplified most molluscs and crustaceans, except C. crangon and I. argentinus. This primer couple is promising to be employed universally for species identification of crustaceans and moluscan taxa. In addition, UCYTB151BF/UCYTB271R and UCYTB152BF/ UCYTB271R were successful to amplify all fish, crustacean and mollusc examined in this study. Hence, these primers can be considered as the first universal primers applicable for fish, crustaceans, and molluscs. However, further trials on a wider variety of species, including species with unusual mtDNA character such as Pectenidae, are necessary to ascertain whether these primers are universally effective to amplify the majority of fish and seafood products. Sequence analysis proved that all validated primer couples were successful in amplifying the expected DNA fragments (~357bp and ~410bp) of cyt b region. BLAST analysis performed similarity indexes against the libraries of 15 selected samples varying between 92% and 100%. Sequencing was also successful to differentiate the 3 unknown sand lances/smelt fish and 2 unknown shrimps. Additionally, RT-PCR was applicable to differentiate between tuna and cod, 2 ray fish (R. clavata and M. senta), 2 crustaceans (P. monodon and P. latisulcatus), and 2 mussels (M. edulis and Perna canaliculus), but failed to discriminate among smelt fish. In order to increase the RT-PCR sensitivity, amplification using HRM Mix is suggested.
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LIST OF ABBREVIATIONS
AFLP: Amplified Fragment Length Polymorphism ATP: Adenosine Triphosphate BLAST: Basic Local Alignment Search Tool BOLD: Barcode of Life Data System COI: Cytochrome oxidase Sub Unit I CTAB: Cetyltrimethyl Ammonium Bromide Cyt b: Cytochrome b ddNTPs: Dideoxynucleotides DGGE: Denaturing Gradient Gel Electrophoresis ELISA: Enzyme-Linked ImmunoSorbent Assay EMBL: European Molecular Biology Laboratory FADH2 : Reduced Flavin-Adenine-Dinucleotide FISH-BOL: Fish Barcode of Life Initiative GE: Gel Electrophoresis IEF: Isoelectric Focusing MALDI-TOF-MS: Matrix Assisted Laser Desorption/IonizationTime of FlightMass Spectrometry NCBI: National Center for Biotechnology Information mtDNA: Mitochondrial DNA NADH: Reduced Nicotinamide-Adenine-Dinucleotide nDNA: Nuclear DNA NMR: Nuclear Magnetic Resonance PCR: Polymerase Chain Reaction PFGE: Pulse-Field Gel Electrophoresis RAPD: Random Amplified Polymorphic DNA RFID: Radio Frequency Identification RFLP: Restriction Fragment Length Polymorphism RT-PCR: Real Time Polymerase Chain Reaction SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SSCP: Single Strand Conformation Polymorphism TGGE: Temperature Gradient Gel Electrophoresis
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LIST OF WEBSITES
BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html BOLD: http://www.barcodinglife.org ChromasPro: http://www.technelysium.com.au/ChromasPro.html DNAsoftware: http://www.dnasoftware.com/Science/primerdesign.html EMBL (EMBL-bank): http://www.ebi.ac.uk/embl/ ExPASy: http://www.expasy.org/ FISH-BOL: http://www.fishbol.org FishTrace: http://www.fishtrace.org Mascot: http://matrixscience.com/ MitoFish: http://mitofish.ori.u-tokyo.ac.jp/ NCBI (GenBank): http://www.ncbi.nlm.nih.gov/ OligoDesign: http://www.invitrogen.com/oligos PeptideMass: http://www.expasy.ch/tools/peptide-mass.html PremierBiosoft: http://www.premierbiosoft.com/primerdesign/index.html PrimerDesign: http://www.primerdesign.co.uk/Biosearch
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APPENDIX I. List of samples used in this study
1. Thunnus sp
2. Eutrigla gurnardus
3. Gadus morhua
4. Trisopterus luscus
5. Solea solea
6. Microstomus kitt
7. Raja clavata
8. Malacoraja senta
9. Ammodytes tobianus
10. Hyperoplus lanceolatus
11. Unknown smelts
P. monodon
M. rosenbergii
13. Illex argentinus

P. latisulcatus C. crangon
12. Crustaceans
M. edulis 14. Perna canalicullus
Mussel-120 Mussel-T1 15. Sea mussels
Mussel-2251
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APPENDIX II. List of chemicals, solutions, and equipment Table II-A. List of chemicals and reagents
Chemical Ethanol p.a. EDTA-Na2 dihydrate Tris Base Nuclei Lysis Solution Proteinase K recombinant PCR RNAase Protein Precipitation Solution Isopropanol p.a. NaOH p.a. HCl 37% PCR-H2O18.2 M cm Company Merck BDH Promega Promega Roche Roche Promega Serva BDH Merck Sartorius Ordering number 8.18760.2500 443885J A5135 A7943 3115879 10109169001 A7953 39559.02 102525P 1.00317.2501 Arium
Table II-B. List of solutions

EDTA 0.5M pH 8.0 93g EDTA-Na2 dihydrate (Mw: 372.2) Add 350ml PCR-H2O, add 10N NaOH until pH 8.0 Add PCR-H2O until 500ml and sterilize for 20 minute at 121C (1 bar) Add 40g NaOH with 100ml PCR-H2O 500ml and sterilize for 20 minute at 121C (1 bar) 30.29g Tris Base (MM: 121.14) with 150ml PCR-H2O in a 250ml volumetric flask. Add HCl 37% until pH 7.4, add with H2O until 250ml and sterilize for 20 minute at 121C (1 bar) Mix 10ml Tris-HCl 1M, pH 7.4 and 2ml EDTA 0.5M, pH 8.0 then add with PCR-H2O until 1 lt. Sterilize for 20 minute at 121C (1 bar). Weigh 100mg RNase A in 25ml PCR-H2O in a 25 ml volumetric flask Weigh 20mg Proteinase K in a 1.5ml microcentrifuge tube. Add 1 ml sterilized PCR water and vortex 10 mM Tris HCl pH 7.5, 125mM NaCl, 10mM EDTA, 0.5 or 1% SDS 48.05 or 72.08g in 200ml TNES buffer
NaOH 10N Tris-HCl 1M, pH 7.4 TE-buffer 1X 10mM Tris-HCl, pH 7.4 1mM EDTA, pH 8.0 RNAse Solution 4mg/ml Proteinase K solution 20 mg/ml TNES buffer with 0.5 or 1% SDS Urea 4 or 6 M
Table II-C. List of equipment

Equipment Plastic tubes Micro centrifuge Filter/fin tip Analytical balance Water bath Vortex Specification 1.5-2.5 ml Sigma 1-14 10 1,000 l 20-110C Mini vortexer-TM1 Company ROTH Sigma Molecular BioProducts Sartorius, Mettler Memmert Techmatic
71

PCR Protocol For Fish and Seafood Authentication MSC Thesis

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ERASMUS MUNDUS MASTER COURSE SEFOTECH.

M.Sc. thesis submitted by: Dwiyitno

M.Sc. thesis submitted by: Dwiyitno

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Gent-Oostende, February 2008 Dwiyitno

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

APPENDIXES Figure I. List of samples used in this study ......................................................................... 70

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

APPENDIXES Table II. List of chemicals, solution and equipment ............................................................ 71

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

CHAPTER II REVIEW OF LITERATURE

RFID movement monitoring (Growing stage)

Barcode label tracking (Processing & distribution)

RFID & Hatchery sampling

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

1D & 2D Gel Electrophoresis

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

-------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ -------++++++++++++++++++ +++++

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

250 275 Wavelength (nm)

600 Wavelength (nm)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

2nd Cycle, etc...

primer primer primer

Separate 2 DNA strands, add primer

Add DNA polymerase

Separate 2 DNA strands, add primer

Add DNA polymerase

Figure 2-5. The principle of PCR amplification

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

CCATCCAACATCTCAGCATGATGAAA CCCCTCAGAATGATATTTGTCCTCA CCATCCAACATCTCAGCATGATGAAA CCCCTCAAAATGATATTTGTCCTCA GTCGAATGAATCTGAGGAGGCTT CCRATTGGGTTGTTTGACCCTGTTTC

Tuna, bonito, mackerel (Rehbein et al., 1995)

558 348 715

(Hsieh et al., 2005) Scombroidae, Istiophoridae (Richardson et al., 2006)

Teleost fish (Sevilla et al., 2007)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

P to be incorporated to the ddNTP. The extended DNA is

Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single Strand Conformation

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

CHAPTER III OBJECTIVES OF PRESENT STUDY

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

3.3. Research framework

Morphological identification (if available) Comparison of DNA extraction methods

DNA quantity and purity

PCR product analysis

- Evaluation of targeted fragments - Index of similarity (BLAST)

- Evaluation of melting points (Tm) - Identification of dissimilarity

: Focus of the study

Figure 3-1. Framework of present study

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

CHAPTER IV MATERIALS AND METHODS

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)