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SEFOTECH.NUT
Dwiyitno
Dwiyitno ou=SefotechNUT,
email=dwiyitno@yahoo.com
Date: 2009.04.11 18:47:54
+07'00'
2008
KaHo-Sint Lieven
School for Engineering
Gebr. Desmetstraat 1
9000 Gent - Belgium
Dwiyitno
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%. RT-
PCR 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
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 Cathólica Portuguesa-
Portugal, 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 Ma’ruf, 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.
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
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) iii
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
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
LIST OF FIGURES
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
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
LIST OF TABLES
V. RESULTS
Table 5-1. DNA concentration and purity yielded by different extraction methods ............... 41
Table 5-2. Optimal annealing temperature (ºC) 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
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) vii
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
CHAPTER I
INTRODUCTION
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
(Céspedes 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
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; Necidová 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 GMO’s 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).
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.
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).
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 organism’s
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 (O’Farrell, 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).
Metagenomics Community
Toolbox
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.
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 animal’s
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).
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, CBOL’s, FISH-BOL, and Fishtrace.
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)
12SrRNA Cyt b
ND6
Mitochondria 16SrRNA
Nucleus ND5
22 tRNA genes
ND1
12 protein-coding regions
ND2 ND4
ND4L
ND3
COI COIII
ATPase6
COII ATPase8
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 Phylum Class Order Family Genus Species
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 +++++
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
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; Céspedes 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.
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
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 low-
cost, 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).
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).
numerous commercial kits are available for isolation of total DNA. The use of commercial
kit permits production of higher quality DNA isolate.
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 μl of TNES consisting of 10-
200 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).
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).
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 55-65°C
(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 37-45ºC, it must be applied separately from Proteinase K.
Likewise, Proteinase K rapidly inactivates nucleases including RNase. Once cellular
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 μl lysis solution was used to extract 50-
100mg of total DNA from fish tissue and fish fin with no addition of RNase, carried out at
37-42ºC incubation for overnight (8-16 hours) (Asahida et al., 1996). Wasko et al. (2003)
examined 75ng/μl proteinase for 100-300mg of fish fin and scales at 42ºC for 10 hours
followed by the same concentration of RNase (42ºC for 1 hour) while Hsieh et al. (2003)
worked with 400ng/μl on fish tissue without RNase. In addition, Imai et al. (2004)
employed relatively high concentration of Proteinase (4000ng/μl) for digesting crab (genus
Scyla) samples at 55ºC for 3 hours followed by 1000ng/μl RNase at 37ºC for 30 minutes.
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.
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.
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.
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 µm in diameter
depending on the type of sample: cells, nucleic acids, or proteins) allow to minimize non-
specific 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.
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).
1.5
DNA
dsDNA Ex:480nm
1.2
260 nm
0.9
OD
0.6
0.3 ssDNA
RNA
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
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 94-95ºC 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 35-60ºC 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 72ºC 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).
primer
primer
primer
DNA template Separate 2 DNA Add DNA Separate 2 DNA Add DNA
strands, add primer polymerase strands, add primer polymerase
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 site-
specific 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 (Céspedes 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).
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 primer-
dimers 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
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:
Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood
Targeted
Name of Size Target species and
Sequence (5’-3’) Fragment
primer (mers) Source of reference
(bp)
L14841 AAAAAGCTTCCATCCAACCAACATCTCAGCATGATGAAA 31 Vertebrate species
AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA 376
H15149 30 (Kocher et al., 1989)
Cyt BL CCATCCAACATCTCAGCATGATGAAA 26 Tuna/family Scombroidae
CCCCTCAGAATGATATTTGTCCTCA 358
Cyt BH 25 (Bartlett & Davidson, 1991)
Cyt BL1 CCATCCAACATCTCAGCATGATGAAA 26 Salmon, mackerel, hering, cod
358
Cyt BH CCCCTCAAAATGATATTTGTCCTCA 25 (Bartlett & Davidson, 1992)
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.
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 72ºC (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).
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.
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.
SybrSafe, SybrGreen, SybrGold (Molecular Probes®) and GelRed, GelGreen (Biotium Inc.)
are established commercially, despite their costs are higher than EtBr.
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 (1000-
2000bp) 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).
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).
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 37°C, 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).
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).
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).
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
Sample collection
CHAPTER IV
MATERIALS AND METHODS
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, FishTrace’s 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)
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:
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; Céspedes 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.
The DNA was extracted by adding 200 μl 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 μl of
isopropanol (IPA). After being chilled at -20ºC for 1 hour, the DNA was precipitated at
14.000 rpm for 10 minutes. The DNA pellet was washed with 600 μl of 70% ethanol, air
dried and resuspended in 100 μl TE buffer.
In a 1.5 ml Eppendorf tube, approximately 100 mg of tissue was crushed with 600
μl TNES buffer (1% SDS). 15 μl of 20 mg/ml Proteinase K was added to each tube
followed by overnight incubation at 55ºC. The DNA isolation was performed by 600 μl 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 μl 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 -20ºC for
1 hour, the tubes were centrifuged at 14.000 rpm for 10 minutes. The DNA pellet was
washed in 600 μl of 70% ethanol, air dried and resuspended in 100 μl of TE buffer.
Approximately 100 mg of tissue was transferred into a 1.5 ml Eppendorf tube. 600 μl
of TNES-Urea buffer (6 M urea; 1% SDS) was used as the digestion buffer. 2μl of 20
mg/ml Proteinase K was added to the tube for protein digestion and the mixture then
incubated overnight at 37ºC. After this period, 3 μl of RNase was added to the tubes to and
the tubes were maintained at 37ºC for 30 minutes.
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 -20ºC 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 μl of TE buffer,
incubated at 65ºC for 1 hour and ready for downstream applications.
700
600
Y = 20.592X + 9.1132
R2 = 0.9993
Relative Fluoroscence
500
400
300
200
Ex : 480nm Em : 520nm
100
0
0 5 10 15 20 25 30 35
DNA (ng/ml)
Sampling arm
Lower
measurement upper
pedestal measurement
pedestal
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 μl of samples were
prepared and to each of them was added one drop of blue-orange loading dye (Promega). A
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 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 20μl. 10μl 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/μl Taq DNA polymerase. 2 μl of each
primer (10mM) and serial dilution (5 and 20 ng/20μl) 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).
Figure 4-6. Software for sequence analysis: ChromasPro (left) and BioEdit (right)
Prior to the RT-PCR amplification, all reagents (2x QuantiTect SYBR Green RT-
PCR Master Mix and primers) were thawn. The master mix was prepared to a final volume
of 20 μl according to the manufacturer’s instructions (Table 4-5). Into each microplate
well, 15 ul of the mix solution were filled. 5 μl of DNA template (final concentration of
10ng/20μl) 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 (60-90ºC
with ramp rate 2.2-4.4ºC/s). The melting points (Tm) generated from the melting curve
(Figure 4-7) were used to discriminate between closely-related species.
Figure 4-7. Typical results of RT-PCR: annealing curve (left) and melting curve (right)
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)
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
ddNTP’s: 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/Ionization–Time of Flight–Mass
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
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
9. Ammodytes tobianus
P. monodon M. rosenbergii