11
Application of Molecular Biology Techniques in Food Forensics

Food forensics is a diverse discipline that covers various techniques and methods throughout all major scientific spheres, such as molecular biology, microbiology, physics, biochemistry, chemistry, etc. Molecular biology techniques involve protein and DNA analysis for the identification, fingerprinting, and authenticity testing of food samples or species presented as evidence. Molecular biology methods rely on the presence of rare features of the species’ genome or peptide sequence at a specific level of taxonomy that is highly dependent on the level of specificity needed as evidence that can be trusted beyond reasonable doubt for identification.

Molecular science methods are known to have a sensitivity high enough to correctly match the specimen found in the food to the corresponding species, even though the food material has been highly degraded during the multi‐stage steps involved in processing or in cases where the sample contains very low quantities of the genetic material. These methods are both qualitative and quantitative, thus attractive for use in food forensics and food forensic‐toxicology. This chapter will focus mainly on the usefulness of molecular techniques in the identification of forensic cases for the types of foods that originate from certain groups of animals and plants. Animal groups will include those from edible species of vertebrates, such as selected mammalian livestock (cows, sheep, swine, and goats), endangered mammalian wild animals (rhinoceroses and tigers), aves or birds (chicken, ostrich, and ducks), and actinopterygian (fishes, e.g. tuna, etc.), as well as edible aquatic turtles.

Introduction

Reports on food forensic cases are many and involve foods from a diverse range of species. However, some species or groups of animals have been more studied than others. According to Teletchea et al. (2005), the frequency of studies for various vertebrates follow the order, mammals > fishes > birds. In plant crops, only a few species have been reported on.

Molecular Biology Techniques in Food Forensics

Techniques in molecular biology may be grouped based on the information that is required and also the type of genetic processes that need to be investigated. Some of these techniques may involve dissecting the genome into manageable‐sized segments for manipulation and sequencing of the DNA, while others require the separation of specific target macromolecules from the mixtures found in the cell. Also, in some cases, genetic analysis may involve the use of tools such as model organisms to substantiate the study and findings. All these methods can be used to qualify the composition and elements of different types of food products and provide the supporting evidence that may be used in forensic cases related to foods.

Molecular biological techniques for species identification and authentication takes advantage of the genetic diversity among organisms for the identification, classification, and authentication of species by estimating species diversity indices. A diversity index represents a measure of species diversity in a population. There are several genetic diversity parameters that may be used for species identification (Begon et al., 1996; Magurran, 1988, 2004; Rosenzweig, 1995; Roth et al., 1994) and they include the following:

Percentage or Proportion of Polymorphic Loci

This parameter provides a measure of the extent of genetic variation within the population under study. It can be computed as the ratio of the number of polymorphic loci to that of the total number of loci that have been investigated, both polymorphic and monomorphic. There are several measures that can give the magnitude of the proportion of polymorphic loci and they include a measure of heterozygosity as well as homozygosity, which are defined as the percentage of individuals observed in each locus. For example, if it can be assumed that N is the sample size in a chosen population, where Nhet is the total heterozygous individuals and Nhom is the total homozygous individuals, then the observed heterozygosity can be calculated as Nhet/N, while the Nhom/N ratio will be for the observed homozygosity. Another measure of the magnitude/extent of the percentage of polymorphic loci is the nucleotide diversity (denoted by the symbol π), which is actually a measure of genetic variation measured in terms of the variation in the DNA sequences. It represents the average number of nucleotide differences in every site in two DNA sequences being compared from the population. A measure of nucleotide diversity has the same measure as that of the average measure of heterozygosity worked out over all nucleotide positions and is mathematically a nucleotide diversity that can be calculated, as shown in Equation 11.1:

(11.1)

where xi is the frequency of the ith sequence and dij is the fraction of nucleotides at which sequences i and j differ.

Generally, it is possible to estimate the measure of nucleotide diversity by either examining the DNA sequences directly, or by making use of molecular marker data.

Shannon Diversity Index (H) and Simpson’s Index (D)

The Shannon diversity index (H) enables the characterization of species diversity within a specific community. Shannon and Simpson’s indices provide a measure and magnitude related to the abundance and measures of evenness of the species present in a population. Mathematically, the Shannon diversity index (H) is calculated as per Equation 11.2:

(11.2)

where i denotes the proportion of species i and p represents the proportion of the total number of species (p _i ).

The higher the value of the Shannon index, the more the richness and evenness within the population under study.

On the other hand, Simpson’s index (D) has values between zero (0) and one (1), and provides a measure of dominance and is inversely proportional to diversity in the population such that if the magnitude of Simpson’s index (D) increases, then diversity in terms of evenness decreases. Simpsonʼs index also provides a measure of the probability that two individuals from an infinitely large population may belong to the same species, and is calculated as shown in Equation 11.3:

(11.3)

where again p _i is the proportion of individuals found in species i.

Jaccardʼs Index

Jaccard’s index, which is important in cases where a comparison of biodiversity levels is required, has to be performed across some geographical sites and is calculated using Equation 11.4:

(11.4)

where S_a and S_b represent the numbers of species unique to samples a and b, respectively, while S_c represents the number of species common to the two samples.

Other parameters for measuring diversity include genetic similarity, F; mathematically, F = 2m_xy/(m_x + m_y), where m_y represents the number of gene fragments shared by both species (X and Y), and m_x and m_y represent the gene fragments by X and Y respectively. F takes values between zero (0) and one (1). Another parameter, known as the genetic distance, is worked out mathematically as 1 – F.

Sampling

For forensic investigations related to the origin of wild animal species (tiger, white rhino, and elephant) and even domesticated animals, the types of sample specimen that are normally collected include hair/fur, muscle tissue, bloodstains, bone, ear, skin, urine, and feces. For food products of animal, fish, and bird origin, appropriate tissues or organs (e.g. blood, meat‐and‐bone‐meal, meat‐meal, skin, bones, tanned hides, fish gills, fins, cartilages, scales, whole organism for smaller species, e.g. sardines, etc.) are taken as sample specimens. These are then processed using various regimes of sample preparation methods utilizing solvent extraction principles of commercially available kits to eliminate unwanted components in the matrix before gene amplification with appropriate primers (e.g. 16S rRNA, cytochrome b, etc.). Table 11.1 summarizes solvent systems used for sample preparation methods and their applications as reported in the literature.

Table 11.1 Preparation of solutions and buffers for molecular analysis (Nishiguchi et al., 2002).

Stock solution	Preparation
Ammonium acetate (Mwt 77.08 g/mol; 5 M) Ammonium sulfate (Mwt 132.14 g/mol; 1 M)	Weigh accurately an appropriate mass of respective salt using an analytical balance and dissolve in small volume of nucleic acid free water in a volumetric flask of a desired volume, and after complete dissolution, make to volume.
5‐bromo‐4‐chloro‐3 indolyl‐phosphate (BCIP; 50 ppb)	Dissolve BCIP in dimethyl formamide to make a stock concentration of 50 ppb BCIP. This stock solution should be kept in freezer at −20 °C.
Buffers: Carlos lysis buffer (100 mL)	Mix the following:‐ Tris (0.1 M, pH 9.5) – by mixing Tris base (1210 mg) dissolved 700 mL of nucleic acid free double distilled water with hydrochloric acid (9.5 mL), 0.2 M ethylenediamine tetraacetic acid (EDTA, 760 mg) and 1.4 M sodium chloride salt (8180 mg). 2% Cetyl trimethyl ammonium bromide (CTAB), 1% polyethylene glycol (PEG‐8000 or 6000 grade, 1000 mg) with stirring for 12 hr. Make up to volume (100 mL in a volumetric flask). NB: β‐mercaptoethanol (2 µl/mL) shouod be added to the buffer prior to use. Mix 1 mM Tris (pH 8.0), 50 MEDTA and 5% (w/v) Chelex‐100 resin.
Chelex‐100 buffer	Mix 0.1 M Tris‐HCl (pH 8.0), 1.4 M sodium chloride, 0.02 M EDTA, 2% (w/v) CTAB, 0.1% (w/v) polyvinylpolypyrrolidine, 0.2% (v/v) β‐mercaptoethanol (for buffer solution which is to be used immediately otherwise, do not add in cases where the buffer is not used immediately).
Cetyl trimethyl ammonium bromide (CTAB) extraction buffer	Mix sodium chloride (0.1 M), Tris (pH 8.0, 0.1 M), and EDTA (0.025 M).
Digestion buffers (aliquots of 10 mL)
Dimethylsulfoxide (DMSO) buffer	Use HPLC grade DMSO. Dispense in smaller volumes (e.g., 1 mL) and store them in a freezer (‐20 °C). NB: DMSO buffer is used once.
Lysis buffer	Mix EDTA (0.1 M), Tris‐HCl (0.01 M, pH 7.5).
Liftons buffer	Mix EDTA (0.1 M), Tris‐HCl (0.025 M, pH 7.5) and sodium dodecyl sulfate (SDS) (1%).
Guanidium isothiocyanate (GITC) homogenization buffer	Prepare the following: 4 M of guanidinium isothiocyanate (Mwt. 118.16); 0.1 M Tris‐HC1 (pH 7.5) and 1% β‐mercaptoethanol by dissolving 50 g of guanidinium isothiocyanate in 10 mL of 0.1 M Tris‐HC1 (pH 7.5) and nucleic acid free water in 100 mL volumetric flask. Heat at 65 °C to facilitate dissolution then filter the solution using Whatman No. 1 or by using a Nalgene Filtration Unit. Alternatively, this solution may be prepared without any need for filtration by using 25 g of guanidinium isothiocyanate in 5 mL of Tris‐HCl, and nucleic acid free water in a sterile 50 mL volumetric flask. NB: – β‐mercaptoethanol should be added to the final concentration of 1% (0.14 M) just before use.
Maleic acid buffer (MAB)	Prepare MAB by mixing 0.1 M maleic acid and 0.15 M sodium chloride, pH 7.5.
[3‐(N‐morpholino)propanesulfonic acid] – (MOPS) buffer	Prepare MOPS by mixing MOPS (0.1 M, pH 7), sodium chloride (0.5 M) and Tween‐20 (0.1%).
Phosphate‐buffered saline solution (10x PBS)	10xPBS is prepared by mixing sodium chloride (80 g), potassium chloride (2 g), of sodium phosphate (14.4 g) and potassium phosphate (2.4 g) and dissolve in 800 mL of nucleic acid free water, adjusting the pH to 7.4 with HC1 and make to volume (1 litre), autoclave and the solution is stored at room temperature.
Phosphate buffered saline, Triton X‐100 (PBT)	PBT is a solution of 0.1% of Tween‐20 in 1xPBS buffer.
10x PCR buffer	Mix:‐ molecular biology grade Tris base (8.116 g), 0.610 g of magnesium chloride (0.610 g) and ammonium sulfate (2.227 g) in HC1 (90 mL), stir to facilitate dissolution, make to volume (100 mL sterilized volumetric flask with H). Autoclave and dispense volumes of 1 mL eppendorf tubes.
PCR buffer w/non‐ionic detergents	This is prepared by mixing potassium chloride (0.05 M), Tris‐HCl (0.01 M, pH 8.3), magnesium chloride (0.0025 M), gelatin (0.1 ppb, i.e 0.1 mg/mL), NP‐40 (0.45%) and Tween‐20 (0.45%). NB: This buffer cannot be filtered or autoclaved, it should therefore be prepared in aliquots of smaller volumes, eg.10 mL in sterile falcon tubes using sterile reagents. For example, prepare using smaller volumes and weights, such as potassium chloride (0.5 mL, 1 M), Tris‐HC1(0.1 mL, 1 M, pH 8.3), magnesium chloride (25 μL, 1 M), gelatin (1 mg), NP‐40 (450 μL), Tween‐20 (450 μL) then make to volume (10 mL) using nucleic acid free water.
PK buffer	PK buffer is prepared by mixing Tris‐HC1 (1 M, 10 mL) and EDTA (0.5 M, 2 mL) and make to volume (200 mL) using nucleic acid free water.
RNA preparation binding buffer	Mix stock solutions of nucleic acid free sodium chloride (0.5 M), TrisCl (10 mM, pH 7.4), EDTA (1 mM, pH 8.0), autoclave, cool to 65 °C then add 0.1% SDS from a 10% stock solution.
RNA preparation elution buffer	Mix nucleic acid free stock solutions of Tris‐HC1 (10 mM, pH 7.4 and EDTA (0.1 mM, pH 8.0), autoclave the solution and cool to 65 °C.
RNA preparation washing buffer	Mix nucleic acid free stock solutions of Tris‐HC1 (10 mM, pH 7.4) and sodium chloride (100 mM) cool the mixture to 65 °C. Then add sodium lauryl sulfate sarcosinate (also called SDS) from a 10% stock solution and EDTA (1 mM, pH 8.0).
Sarkosyl buffer (5%, 500 mL)	Prepare by dissolving sarkosyl (25 g) in sodium chloride (15 mL, 5 M), Tris‐HC1 (25 mL, 1 M, pH 8.0), EDTA (15 mL, 0.5 M) and 400 mL of H then make to volume (500 mL sterile volumetric flask) using nucleic acid free water. NB: Do not refrigerate or autoclave this solution.
SDS buffer (4%)	Dissolve sodium dodecyl sulfate (20 g) in sodium chloride (30 mL, 5 M), Tris‐HCI (25 mL, 1 M, pH 8.0), EDTA (100 mL, 0.5 M) and nucleic acid free water (300 mL).
Sodium Chloride‐Tris‐EDTA buffer (STE)	STE is prepared by mixing sodium chloride (0.1 M), Tris‐HCI (10 mM, pH 8.0) and EDTA (1 mM, pH 8.0).
50x Tris‐Acetate Buffer (TAE)	50x TAE is prepared by dissolving Tris base (242 g) in glacial acetic acid (57.1 mL) and EDTA (100 mL, 0.5 M pH 8.0) in nucleic acid free water and make up volume (1 litre). NB 1: The 50x TAE is a highly concentrated stock solution. NB 2: Dilute this solution to make lx TAE (0.04 M Tris‐acetate, 0.001 M EDTA) and use it as your working solution.
5x Tris‐Borate/EDTA buffer (TBE)	Dissolve 54 g of Tris base, 27.5 g of boric acid, and 20 mL of 0.5 M EDTA (pH 8.0) in H2O up to 1 liter. Stir until dissolved. The 5x TBE is the concentrated stock solution. Use 0.5x TBE (0.045 M Tris borate, 0.001 M EDTA) as electrophoresis buffer. NOTE: A precipitate forms when concentrated solutions of TBE are stored for long periods of time. Discard any batches that develop a precipitate. NOTE: 10x TBE buffer is commercially available, and it constitutes a good solution for laboratories not using much TBE buffer, or for laboratories with high budgets.
TBST solution	135 mM NaCl 2.7 mM KCl 25 mM Tris HC1 (pH 7.5) 0.1% Tween‐20 2 mM levamisole (add on day of use).
TE Buffer Solution (pH 7.4)	10 mM Tris‐HCl, pH 7.4 1 mM EDTA, pH 8.0
TE Buffer Solution (pH 7.60)	10 mM Tris‐HC1, pH 7.6 1 mM EDTA, pH 8.0
TE Buffer Solution (pH 8.0)	10 mM Tris‐HCl, pH 8.0 1 mM EDTA, pH 8.0
CTAB + NaCl solution (10 % : 700 mM)	Mix CTAB (appropriate weight, e.g 10 g for 100 mL stock volume) and 700 mM sodium chloride (appropriate volume), heat with stirring to dissolve, make it to volume (eg., 100 mL, 1000 mL etc).
H‐treated diethyl pyrocarbonate (DEPC)	Dissolve 0.5 % DEPC in H, then homogenize by thoroughly stirring in the fume cabinet then allow the solution to sit for sometime then autoclave. NB: Composition for HM include calcium chloride (1 mM), sodium bicarbonate (1.5 mM), magnesium chloride (0.1 mM), magnesium sulfate (0.08 mM) and potassium nitrate (0.03 mM) all dissolved in Arrohead spring water.
Dodecyltrimehylammonium bromide (DTAB)	Mix 8% DTAB with sodium chloride (0.015 mM) and Tris (100 mMn pH 8.8) and EDTA (50 mM).
Ethylene diamine tetraacetic acid (EDTA)	Mix disodium ethyl anediaminetetraacetate (168.1 g) with H (800 mL) with thorough stirring, adjust to pH 8 using NaOH (or instead add ~ 20 g NaOH pellets) in order to enhance complete dissolution of EDTA, then autoclave the solution.
Ethidium bromide (10 ppb)	Mix ethidium bromide (0.2 g) and 20 mL of H (refer above), stir to dissolve the dye and then store at room temperature in the dark room wrapped in an aluminium foil.
Glycine (2 ppb)	Dissolve 2 mg in 1 mL PBT (phosphate buffered saline, Triton X‐100) then store in a freezer at −20 °C. NB 1: PBT is prepared by dissolving 0.1% Tween‐20 surfactant in PBS (phosphate buffered saline solution). NB: 2: PBS is prepared by dissolving sodium chloride (80 g), potassium chloride (2 g), sodium phosphate (14.4 g), and potassium phosphate (2.4 g) in a sufficient amount of nucleic acid free water (e.g., 800 mL) then ajust the pH of the solution to 7.4 using HC1. Make to volume using nucleic acid free water (in a 1 litre sterile volumetric flask). Autoclave the solution and store the solution at room temperature.
Guanidium thicyanate (5 M)	Mix guanidium thiocyanate (59 g) with nucleic acid free water (in a sterile 100 mL). Heat at 65 °C to dissolve the salt. Filter the solution using Whatman (No 1, or using Nalgene filtration unit).
Lithium chloride (4 M)	Prepare by dissolving an appropriate amount of lithium chloride (Mwt, 42.39 g/mol), e.g 169.56 g in an appropriate volume of nucleic acid free water then make it to 1litre (steriled volumetric flask).
Levamisole (1 M)	Preapare this solution by dissolving levamisole (0.06 g) in 0.25 mL of nucleic acid free water. This solution should be prepared only when is needed to be used (should not be stored!).
Magnesium chloride (1 M)	Prepare by dissolving an appropriate weight of magnesium chloride hexahydrate (Mwt 203.31) in an appropriate volume of nucleic acid free water and make it to volume (eg 100 mL, volumetric flask, 1 L, etc). For example use 203.31 g of MagCl2 and 800 mL of water the make it to volume (1 L) with water. NB: Magnesium chloride is highly hygroscopic, do not store and also do not open the bottles for a long time.
4‐nitroblue tetrazolium chloride (NBT)	Prepare NBT solution by dissolving 75 mg/mL dimethyl formamide (70%). Store in a freezer at −20 °C.
NTMT solution	Prepare NTMT by mixing sodium chloride (0.1 M); Tris‐HCl (0.1 M, pH 9.5) magnesium chloride (0.05 M) and Tween‐20 (0.1%). When using NTMT solution add 0.002 M levamisole at that time.
Paraformaldehyde (4 %)	This solution prepared by dissolving paraformaldehyde (10 g) in DEPC‐treated H (200 mL), heated at 65 °C in a fume hood and then cooled on ice. Then using NaOH (5−10 µl) correct the pH to 7.5. Add 10x PBS (25 mL) and make volume up to 250 mL with DEPC treated nucleic acid free water then store the solution at −20 °C.
phenol:chlorophora:isoamyl alcohol (PCI)	The PCI solution is prepared by mixing phenol, chloroform, and isoamyl alcohol, at a ratio of 25:24:1. at a pH of 7.5−8.0. NB: For molecular analyses, a commercial PCI may be better.
Potassium acetate (5 M)	Prepare by dissolving potassium acetate (49.1 g, Mwt 98.15) in 90 mL of nucleic acid free water then adjust the pH to 7.5 using 2 M acetic acid. Make to volume (100 mL) and the solution can be stored in a freezer at −20 °C.
Proteinase K (20 mg/mL)	Prepare proteinase K by mixing Tris (pH 7.8), EDTA (0.005 M) and 0.5% sodium dodecyl sulfate (SDS), incubate the solution at 37−56 °C and then store Proteinase K at −20 °C. NB: Proteinase K should be used at a concentration of 50−60 µg/mL with a reaction buffer containing 0.01 M of the prepared solution (Tris + EDTA + SDS).
10% SDS (100 mL)	Prepare by dissolving sodium dodecyl sulfate (10 g) in nucleic acid free water and then make it to volume (in 100 mL sterile volumetric flasks).
20x SSC (pH 4.5)	Prepared by mixing sodium chloride (3 M) and sodium citrate (0.3 M).
Silica solution	Prepare by dissolving silica dioxide (4.8 g) in nucleic acid free water (40 mL) in a polypropylene tube of appropriate volume, then homogenize by agitating the tube with contents. Allow it to stand 24 hrs then dispense 35 mL accurately (using a pipette), add 5 mL to make a volume of 40 mL with nucleic acid free water (or distilled water), agitate and allow this solution to stand for 5 hrs. Pipette 36 mL and add to it 48 mL of HCl. Dispense smaller volumes of 1.5 mls and store in the dark room.
3 M sodium acetate (NaOAc) (pH 5.2 and 7.0)	Dissolve 40.82 g of sodium acetate trihydrate (CH3COONa•3H2O; M.W. 136.08) in 80 mL of H2O. Adjust the pH to 5.2 with glacial acetic acid or adjust the pH to 7.0 with dilute acetic acid. Adjust the volume to 100 mL with H2O. Sterilize by autoclaving.
2 M sodium acetate (pH 4.0)	Dissolve 27.22 g of sodium acetate trihydrate (CH3COONa•3H2O; M.W. 136.08) in 80 mL of H2O. Adjust the pH to 4.0 with glacial acetic acid. Adjust the volume to 100 mL with H2O. Sterilize by autoclaving.
5 M sodium chloride	Dissolve 292.2 g of sodium chloride (NaC1; M.W. 58.44) in 800 ml of H2O. Adjust the volume to 1 liter with H2O. Sterilize by autoclaving.
Solution I	50 mM glucose 25 mM Tris‐HC1 (pH 8.0) 10 mM EDTA (pH 8.0) Solution I can be prepared in batches of 100 mL. Autoclave for 15 minutes on liquid cycle. Store at 4 °C.
10 mM Tris‐HC1 (pH 7.4, 7.6 and 8.0)	Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to the desired value by adding concentrated HCl. pH 7.4 add 70 ml. pH 7.6 add 60 ml. pH 8.0 add 42 ml. Other pHs desired can be obtained by titrating the HCl. Adjust the final volume of the solution to 1000 ml with H2O.

Preparation of the Genetic Materials

The preparation of genetic materials involves three main steps. The first step involves the preparations of buffers and solutions needed in the molecular analyses, while the second step deals with the extraction of the nucleic acids; the third step involve the purification of the genetic material to ensure that it is of high quality. Normally, when the nucleic acid material has been extracted and purified, it is re‐suspended in either distilled water or TE buffer solution (1 µg nucleic acid/μL).

Preparation of Common Solutions for Molecular Analysis in Food Forensics

Sample preparation also involves the extraction and purification of the gene (DNA fragment) for molecular identification and fingerprinting purposes. The DNA extraction and molecular analysis requires the use of some solutions, which can be prepared in the laboratory. Some of the most common solutions are displayed in Table 11.1.

Molecular Methods for Genetic Materials Extraction

Molecular methods for genetic materials extraction can be carried out by using commercially available kits. These kits can be obtained from various vendors/companies that offer various commercial kits such as QIAamp® DNA Mini Kit #51304, #51306; DNeasy® Tissue Kit #69504, #69506, etc.; Nucleospin® (http://www.clonetech.com), Isoquick® (Microprobe Corp.); and Bio 101® (http://www.bio101.com). Commercial nucleic acid extraction kits contain reagents and solutions required for nucleic acid extractions. For example, commercially available kits may include enzymes such as proteinase K, extracting buffers such as non‐phenol/chloroform buffers, and purification columns such as silica‐gel membrane spin‐column, as well as extraction protocols that have been simplified.

DNA Marker Fragments

There are several targeted DNA/genetic markers that are normally used for species identification, as well as other food forensic‐related analytical applications. They include cytochrome (Cyt) b gene (mainly mitochondrial cyt b genes), 16S rRNA, 12S rRNA, 5S rRNA, D‐LOOP, ATPase, COI, ND3/ND4, ATPase 8, NADH2, ND4, etc.

Sample Preparation Methods Suitable for Molecular Biology Techniques

These are summarized in Tables 11.2 and 11.3.

Table 11.2 Sample preparation methods for the molecular forensic analysis of freshwater and marine animal food products (Taggart et al., 1992).

Solvent system	Targeted DNA marker fragment and species
Chloroform, methanol, water	Cyt b gene (171 bp); specimen from canned tuna fish (Terol et al., 2002)
Chloroform, phenol, isoamyl alcohol + Molecular biology grade Chelex resin	Cyt b gene (158 bp) specimen from canned sardines (Jérôme et al., 2003)
Phenol‐Chloroform	– Cyt b gene (875–876 bp) – Sea turtle (muscle, blood, eggs, skin) (Moore et al., 2003)
Phenol‐Chloroform	– Cyt b gene (155–188 bp) – Shark (soup, dried fin, cartilage pills) (Yan et al., 2005)

Table 11.3 Nucleic acid extraction methods and the target specimens for food forensic molecular analyses.

Forensic specimen, target species and fragment	Recommended Extraction protocol
Dried/salted/unfrozen strips of whales, with the target fragment being the D‐loop	Miscellaneous
Embedded human tissues, which are formalin/paraffin fixed, with the target fragment being microsatellites, amelogenin	Chelex
Sea turtle muscle, blood, eggs, and skin, with the Cyt b being the target fragment	Phenol‐chloroform
Shark soup, dried fins, and cartilage gills, with Cyt b being the target fragment	Phenol‐chloroform
Tuna canned products, with Cyt b being the target fragment	Chloroform, methanol, water
Sardines canned products, with Cyt b being the targeted fragment	Chelex, phenol, chloroform and isoamyl alcohol
Beef, mutton, pork, chicken meat/bone meal, with tRNA, ATPase being the targeted fragments	Guanidium thiocyanate

Schemes 11.1 to 11.8 also summarize sample preparation methods.

Scheme 11.1 An example of the phenol‐based protocol for DNA extraction.

Scheme 11.2 Protocol for crude total cellular miniprep.

Scheme 11.3 An example of a cesium chloride gradient protocol for the separation of nuclear DNA and organellar DNA.

Scheme 11.4 The use of Chelex as chelating resins for vertebrate DNA extraction.

Scheme 11.5 Formalin‐based procedure for the isolation of DNA from museum‐preserved specimens.

Scheme 11.6 Procedures for the extraction of enriched cytoplasmic nucleic acid from animals.

Scheme 11.7 Protocol for DNA extraction from bird’s tissues and feathers.

Scheme 11.8 DNA extraction from fish tissues.

This protocol can take at least 3 hours and is suitable in cases where large sample size from individual samples need to be investigated for either systematic or population genetic investigations.

This protocol can take up to 72 hours. The other steps for obtaining high molecular weight nucleic acids using phenol‐chloroform‐based approaches have been omitted. If they are to be included, the time taken for the procedures will be even longer.

This procedure can take about 15 min. The chelating resins are known to denature proteins as well as remove that tend to be inhibitory, which known contaminants are. The procedures can also work well with different types of tissue matrices, including blood, liver, and muscle.

This procedure can take up to 24 hours. The specimen (a known amount), can either be minced meat tissue, ground meat tissue, powdered tissue, or dounced tissue and then a known volume of homogenized buffer is added. The preceding procedures are outlined in the scheme below.

This procedure is suitable for a fish specimen, including normal fish tissue, fin snips, and eggs.

The procedure can take up to 3 hours

Requirements:

• ultraviolet hood;
• sterilized razor blade;
• negative extraction controls should be included;
• buffer ATL, AL, AW1, AW2, AE (included in the kit); and
• proteinase K (included in the kit).

DNA Extraction Procedure

• a known amount of avian tissue specimen or feather is placed in a sterilized vial and then ATL buffer is added together with proteinase K (the ratio of these can be roughly 9:1, ATL Buffer:Proteinase K);
• the set‐up is then incubated by either shaking or rotating in an incubator at 55°C until complete dissolution of the sample is achieved. NB: Depending on the situation, the Buffer ATL and Proteinase K may be scaled up, but with proportional ratios without violating the original balance of the two;
• after complete dissolution, the setup can again be incubated at 65°C for about 15 minutes and 95–100% ethanol added to it with vortexing to thoroughly mix and homogenize before incubation at 4°C for 60 minutes; then spin the sample that is placed in a spin column at 8000 rpm for 1 minute and decant the filtrate;
• add AWI buffer (~500 microliter) and centrifuge at 8000 rpm for 1 minute and again decant the filtrate;
• repeat this step using Buffer AW2 and centrifuge at 8000 rpm for about 3 minutes, then discard the filtrate. Introduce preheated (70°C) Buffer AE (~50 microliter) into the sample contained in the spin column and incubate at room temperature for about 45 minutes, centrifuge at 8000 rpm for 3 minutes;
• store at 4°C if the nucleic acid is to be used immediately, or at –20°C if to be used later.

Sample Preparation Methods

In most cases, sample preparation is essential in the majority of analytical and molecular procedures. In isolation of microorganisms from cultures, normally culture dependent methods are used in order to obtain pure cultures of the same species before the identification using phenotypic or genotypic techniques. Some of these sample preparation methods for microbial isolation involve dilution in media such as isotonic buffers, for example phosphate‐buffered saline (PBS), homogenization, and then plating in either adequate selective or differential culture media.

There are also culture independent methods that do not require any pre‐microbial isolation procedures, because the sample/specimen matrix containing the microbial species under investigation will form the sample. The genomic DNA of the microorganisms present in the sample can be extracted using commercially available kits.

Molecular Markers in Food Forensics

Molecular markers refer to the genomic fragments resulting from PCR amplification of random segments of the DNA using a single primer of the target nucleotide sequence. Molecular markers in food forensics play crucial roles in a number of areas, for example, in genetic fingerprinting of both plant and animal varieties, determination of similarities among various varieties, mapping of plant and animal genomes, as well as in the ascertaining of phylogeny among species. Molecular markers enable species and/or their products to be compared in a number of molecular techniques such as through the use of restriction fragments techniques, techniques that rely on the identification of isoenzymes (protein/gel electrophoresis), as well as those that utilize products of the polymerase chain reaction (PCR).

General PCR Procedure

In performing PCR analyses, the PCR mixture is normally prepared to contain an enzyme (PCR polymerase), nucleotide (dNTPs), PCR buffer, and appropriate forward and reverse primers, which may either be genus‐ or species‐specific. The DNA extract is then added to this PCR mix and also for control, NA free water is added to the PCR mix.

The prepared samples (sample and control) are introduced to a PCR machine, and a PCR program is run using optimal conditions, depending on the type of DNA polymerase that was used in the experiment, as well as an optimal annealing temperature suitable for the set of primers that were used. When the program has run to completion, DNA loading buffer is added and then the amplified products are visualized in an agarose gel to which an ethidium bromide stain has been added.

PCR Procedure and Principle

The polymerase chain reaction employs two oligonucleotide primers to hybridize to opposite nucleic acid strands and therefore flank the target DNA sequence being amplified. The process uses enzymes such as Taq polymerase to act as catalysts to enable the elongation of the primers and repetitive series of cycles involving several steps such as: (i) template denaturation; (ii) primer annealing; and (iii) extension of the annealed primers with the ends of the fragment characterized by the 5’ ends of the primers, so that the primer extension products synthesized in a given cycle can also be used as templates in the next cycle.

The manipulation of PCR technology has resulted in an increased use and application to many other different molecular biology products. For example, when the PCR is merged with reverse transcription, it makes it possible to obtain an extended technique RT‐PCR, which can enable the analysis of RNA in addition to DNA. The use of short primers enables the generation of a genomic fingerprint, which is useful in the investigation of genetic information of organisms whose genomic sequences are completely unknown and this is the bottom line of molecular techniques, such as random amplified polymorphic DNA (RADP) and Differential Display. In some cases, molecular tags can be introduced into the PCR products. These molecular tags, which include digoxigenin (DIG), biotin‐labeled dUTP, etc., make the PCR technique very useful for applications in medical diagnostics, since these labeled PCR products can serve as hybridization probes that can detect very small amounts of pathogens.

Genome Segmentation and Identification/Typing Methods: Molecular Techniques

Genome segmentation and identification/typing methods are molecular biology techniques that are useful in food forensic analyses. There are numerous genome segmentation/molecular techniques and approaches, with the majority being polymerase chain reaction (PCR)‐based methods (classical and improved classical PCR techniques) that are known to be frequently used in food forensics qualification and quantification of food elements. These methods include amplified fragment length polymorphism (AFLP)‐PCR; inter‐sequence simple repeat (ISSR)‐PCR; simple sequence repeats (SSR)‐PCR; real‐time (RT)‐PCR, sequencing and blotting methods; DNA micro‐array; PCR‐restriction fragment length polymorphism (RFLP); PCR‐forensically informative nucleotide sequencing (FINS); DNA hybridization; PCR‐random amplified polymorphic DNA (RAPD); real‐time PCR; species‐specific PCR primers; DNA sequencing; DNA microarray; and PCR‐specific primers.

Amplified Fragment Length Polymorphism (AFLP)‐PCR Methods in Food Forensics

The amplified fragment length polymorphism (AFLP)‐PCR method is a technique based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases. It involves four steps, which involve:

1. Digestion: using mainly two different restriction endonucleases, whereby one of these enzymes is a 4‐base cutter (e.g. MseI with sequence 5’TTAA3’) and the other is a 6‐base cutter (e.g. EcoRI with sequence 5’GAATTC3’);
2. Adaptor ligation: whereby two different adaptors are used. It should be noted that adaptors refers to short double stranded DNA sequences with sticky ends, which are ligated to the digested fragments such that one adaptor complements to the Msel cut end, with the other to the EcoRI cut end;
3. Amplification process: whereby the DNA fragments with MseI‐EcoRI ends are selected as the DNA template for amplification using two PCR primers, which are complementary to the two adaptors employed in the amplification process and these adaptors are labeled using either radioactive or fluorescence dye to facilitate the detection of DNA bands on the gel. Generally, the number of DNA bands detected by AFLP is normally very high and to reduce this number, it is customary to add certain selective bases (mainly 1‐3 nucleotides) at the 3’‐end of the PCR primers; and
4. Electrophoresis: for the separation of DNA fragments.

In AFLP, which is known to be highly polymorphic, DNA variation is detected based on either the presence or absence of DNA bands due to:

1. the presence or absence of restriction sites; and
2. additional bases (insertion) between two restriction sites that are too large.

Generally, the AFLP‐PCR technique is used to identify and detect the genetic natural variation due to single nucleotide polymorphism (SNP) within the biological specimens (population). All organisms are built by small entities known as cells, which contain a biological molecule known as deoxyribonucleic acid (DNA) encoded with the blueprint for the growth characteristics, composition, and functions or characteristic information of all components that form that particular organism. The various body functions of an organism are encoded in a segment of the DNA known as a gene (e.g. information about the structure and sequence of a particular protein or enzyme, height or skin/hair/eye color, etc.). The sum of all genes in an organism is collectively known as a genome. However, organisms that belong to the species have their DNA identical to about 99.9% with 0.1% difference caused by natural variations within the species in the population, which is here referred to as SNP.

The SNP variations can be detected using AFLP‐PCR technology, which can also locate points of polymorphism simultaneously in an organism’s genome. The AFLP‐PCR technology can thus be used to authenticate the origin, composition, adulteration, diversity, and fingerprinting in food forensics. AFLP‐PCR can also be instrumental in detecting illnesses that are foodborne within a particular food chain. It can also find application in areas of genetically modified animals/organisms (GMA/GMOs). The PCR technique that is linked to AFLP plays an important role in amplifying the gene, because in most circumstances, the DNA material presented as the sample specimen is always in minute quantities and thus the need for amplification. During the AFLP‐PCR process, a typical fingerprint pattern of various fragment sizes is observed on a gel. These fragments are then matched with an AFLP marker to establish either the presence or absence of the pattern of interest in a particular gene in the sample. In most cases, AFLP‐PCR is performed using restriction enzymes and selective nucleotides, depending on the samples.

The AFLP‐PCR procedures involve cutting the DNA of the samples at specific sites using two restriction enzymes, then this step is followed by ligating the double stranded nucleotide linkers that link the two DNA strand ends. Then the fragments are amplified using PCR before separation and identification using electrophoresis techniques. Then the gel is stained to enable the visualization of the DNA fragments, which can be observed as bands representing particular sizes of the DNA fragments.

AFLP‐PCR Technique

AFLP‐PCR technique is attractive, mainly because there is a synergy between restriction fragment analysis of genetic materials, which is known to be highly reproducible with the capabilities of the PCR technique. The synergy has an advantage of enabling the random and simultaneous analysis of several loci within the genomic materials under study. The principles of AFLP take advantage of the selective nature of the PCR technique on the restriction fragments arising from the digestion of genomic DNA (Zabeau and Vos, 1993). In this method (Scheme 11.9), restriction enzymes (e.g. HindIII, MseI, EcoRI, etc.) are first used to digest small quantities of highly purified genomic DNA and the process is followed by the ligation of the doubly stranded oligonucleotide adaptors to the sticky ends of both ends (5′ and 3′ endings) DNA fragments that result from the digestive action of the restriction enzymes. These DNA fragments that have been ligated are then subjected to a double PCR amplification process under an excessively stringent environment, in a process that involves the use of a complementary set of: (i) primers to the adapter and restriction site sequence; and (ii) an added nucleotide at the 3′ sticky ending of the DNA sequence. The last step will now be used to identify the extent and nature of polymorphisms by subjecting the amplified fragments to denaturing polyacrylamide gel electrophoresis (PAGE) or any other similar technique (Blears et al., 1998; Mueller and Wolfenbarger, 1999).

Scheme 11.9 Summary of the AFLP‐PCR procedure.

Summary of AFLP‐PCR Process

• extract DNA sample from the tissue/specimen. The important requirements for the DNA needed for AFLP is that it must be very clean and of high molecular weight;
• using the appropriate restriction enzyme, ligate the DNA core sequence adapters. In cases where the site to be cleaved is known, the site is denoted with a ^ mark, e.g. HgaI/MboII cleaves the sequence at (5/10) site. Then pre‐amplify the sequence using correct primers and an appropriate nucleotide (N) of interest and the sequence will be as follows:
  • 5'ATGCANNNNN^3’
  • 3'TACGTNNNNNNNNNN^ 5’;
• perform selective amplification using the same set of primers and increase (e.g. triple) the number of nucleotides from the previous step; and
• then separate using gel electrophoresis.

Gel Electrophoresis Separation of Biomolecules and PCR Products

Electrophoresis is a separation technique where its mechanism is based on the transport of charged analytes under the influence of an electric field. This technique is applied in molecular biology to separate biomolecules such as nucleic acids and proteins, since they are charged. Normally, nucleic acids are subject to electrophoresis in either neutral or basic buffers, in order to make them negatively charged (anions) where phosphate groups become negatively charged. Under acidic conditions, these polynucleotides become insoluble in water and thus cannot be separated using electrophoresis. In this modern age, the majority of electrophoresis methods for the separation of biomolecules (nucleic acids or proteins) tend to employ solid supports, for example polyacrylamide gel and agarose gel, which act as anticonvective agents to create an environment that will retain the integrity of separated sample products by lessening the convective transport and the diffusion phenomena, which will cause the appearance of sharp bands (zones) throughout the electrophoretic run. Moreover, these solid supports (gels) play an important role in acting like molecular sieves, which separate the products based on their molecular sizes.

Types of Electrophoresis Methods

For separation of biomolecules, two types of electrophoresis are known: (i) slab gel electrophoresis; and (ii) capillary electrophoresis (CE), which is further subdivided into several modes, mainly capillary zone electrophoresis (CZE) that separates biomolecules on the basis of charge to mass ratio and is normally used for the separation of bases, nucleotides, nucleosides, investigation of damage to DNA, and also the analysis of small‐sized oligonucleotides. Other CE modes are micellar electrokinetic capillary chromatography (MECC), which separates biomolecules based on charge to mass ratio by partitioning into micelles; capillary isotachophoresis (CITP), which separates on the basis of moving boundaries or displacement and is mainly useful as a preconcentration and enrichment method for CZE and MECC. Another CE mode is capillary isoelectric focusing (CIEF), in which separation is dependent on the isoelectric point of the analytes (used mainly for separation of proteins). There is also another CE mode known as capillary gel electrophoresis (CGE), which works as a molecular sieve and on the principles of reptation. CGE is mainly useful in the analysis of oligonucleotides, primers, probes, PCR products, ascertaining of point mutations, and for the sequencing of DNA, etc.

Slab Electrophoresis: (Gel Electrophoresis) – Polyacrylamide (PA) and Agarose

There are a number of variants of the slab gel techniques, which utilize different types of media, mainly polyacrylamide and agarose for the separation of proteins and nucleic acids. These variants include formats such as horizontal, vertical slab gels, and cylindrical rods (Andrews, 1986; Rickwood and Hames, 1983). Unlike in the case of capillary electrophoresis, where separation and detection processes are synchronized through real time analysis, the processes in many of the slab electrophoresis techniques are such that after electrophoresis of the proteins or nucleic acids, the gel matrix is soaked in the fluorescent dye solution (e.g. ethidium bromide solution), washed, and bombarded with ultra violet radiation in order to generate a photograph that shows the fluorescing DNA band patterns with their molecular weights (base pairs) identified with an appropriate marker. Apart from this fluorescence detection technique, autoradiography has also been used for the detection and identification of genetic materials for samples submitted for forensic crime investigations or samples collected from genetic materials that are suspected to be the cause of a certain disease, where specific sequences of the genetic materials in the sample can be identified in order to prove their identity. An example of autoradiography is a technique known as Southern blotting, which was invented by Southern in the mid‐1970s (Southern, 1975), which makes use of DNA probes that are radio‐labeled to target specific complementary DNA fragments in the sample presented for analysis or fingerprinting. Despite the fact that autoradiography slab gel techniques are unpopular, because they are time‐consuming, they still have some attractive features, such as the possibilities for the simultaneous analyses of samples.

Types of Slab Gels Used in Electrophoresis of Biomolecules

The three most used gels in electrophoresis of proteins and nucleic acids are polyacrylamide gel (prepared by the cross‐linking of acrylamide with N,N'‐methylenebisacrylamide), agarose gel (a polysaccharide prepared algae extract known as agar‐agar), and pulsed‐field gel. The choice of the gel to be used is in most cases governed by the size of the biomolecule (nucleic acid or protein) to be analyzed. When dealing with relatively shorter or smaller fragments of nucleic acids or short peptides, then polyacrylamide gels becomes the convenient choice. In this case, sodium dodecyl sulfate (SDS) with polyacrylamide gel electrophoresis (PAGE) is used (SDS‐PAGE). On the other hand, when the size of the nucleic acid is relatively large, a gel with larger pore size becomes the best choice and therefore agarose becomes the gel to be used in this case. During the gelling process, unlike polyacrylamide, agarose does not cross‐link such that dilute 0.2% or more concentrated 0.8% agarose gels can be enough to make a gel set (stiffen) to enable the separation of nucleic acid materials of molecular weights from 150 million and 50 million respectively (Schwartz and Guttman, 2016). Both polyacrylamide and agarose gels are commonly done on DC electrophoresis fields and can separate nucleic acid fragments of size up to 30 kb (Schwartz and Guttman, 2016). When dealing with nucleic acid materials larger than 30 kb (e.g. chromosomal DNA, which has sizes measuring up to 6 Mb), alternating current (ac) electrophoresis fields are normally used and therefore pulsed‐field gel electrophoresis (PFGE) is the preferred gel to use.

Capillary Electrophoresis (CE)

Unlike the slab gel electrophoresis technique, capillary electrophoresis (CE) employs two types of driving forces: (i) it is associated with the force causing the electrophoretic migration; and (ii) it uses force generated by the electro‐osmotic flow (EOF) through the tiny capillaries.

Separation of Biomolecules Using Non‐electrophoresis Techniques

There are other techniques, apart from electrophoresis, that are used to separate biomolecules. These methods can be categorized based on the mode and principles of separation that are used. There are those techniques that take advantage of the chemical and biological properties of the biomolecules to be separated. Methods that fall into this category include immuno‐adsoption‐based methods in which nucleic acids can be separated using immunosorbent or silica adsorption that enables nucleic acid molecules to bind to the stationary phase (silica) surfaces, which also contain specified salts and are at optimized pH conditions. Another category of these non‐electrophoresis techniques utilizes differences in the sizes of biomolecules to separate the analytes. These methods include centrifugation driven by centrifugal force, whereby differences in terms of the measure of retardation occurring during the transport process as electrophoresis is proceeding, creates an environment where there are differences in transport velocities of the analytes, thus effecting the separation. There are also those methods that take advantage of the electrical charge differences between biomolecules to effect their separations.

High performance liquid chromatography (with a variety of detectors, such as fluorescence, UV, etc.) can be used as an alternative to electrophoresis.

Application of AFLP‐PCR in Food Forensics

There are many areas where AFLP may be applied. These areas include poisonous microorganisms that may be introduced into foods of both animal (through injections, feed, water, etc.) and plant origin (through spray, fertilizer/manure, watering, etc.)

Genotypic Detection and Identification Methods: AFLP‐PCR

AFLP‐PCR has been used to trace the presence of type C botulism found in bovine carcasses to the feed (silage) fed to the animals (Myllykoski et al., 2009). The technique was successfully used to identify type C bovine botulism after isolating Clostridium botulinum type C in bovine liver samples and performing the identification procedures, which employed the use of amplified fragment length polymorphism (AFLP). In this work, the results proved that the microorganisms belonged to group III C. botulinum after diagnosis using AFLP‐PCR. The results prompted the call to propose a measure to prevent the botulinum compounds, which are known to be neurotoxic in the animal feed, by introducing an acidification step during the production process for silage. Though this example in its presentation may not be typical of a forensic case, it may turn out to be so if the owners of the farms had been required by law to include the acidification of silage before feeding it to animals and if it had been proved that they deliberately bypassed this step for economic reasons or otherwise.

Identification of Toxigenic Strains in Animal Carcasses and Varieties in Plant Crops

There have been cases where meat carcasses were deliberately contaminated with toxic substances released by certain strains of toxigenic microorganisms, by either avoiding or deliberately omitting appropriate procedures that are meant to eliminate such microorganisms, with the intention of avoiding costs or to intentionally cause harm to consumers. These microbes may be derived from either water or animal feed, in which the steps to inactivate or kill pathogenic microorganisms are omitted, either to save on production costs or to intentionally cause damage to people’s health (intentional crime). When these animals are slaughtered, the meat products tend to contain the toxic compounds released by microorganisms and if they are not detected before consumption, may affect the health of consumers. This may turn out to be a legal case, as consumers are likely to sue meat vendors, butchery owners, owners of abattoirs, and animal farmers. This situation may necessitate and call for the tracing of the source of the problem, as well as the culprits.

Another example where the AFLP method was applied in plants, where leaves of certain cultivars of grapevines were artificially infected with the fungi species (oomycete Plasmopara viticola) to study the molecular compatibility vis‐à‐vis disease development (Polesani et al., 2008). The cDNA‐AFLP was successfully used to selectively amplify more than 100 primer combinations, enabling the identification of thousands of transcript‐derived fragments (TDFs) from the fungal infected leaves. After sequencing some of these fragments, about 82% were attributed to the grapevine cultivar, about 10% were correlated to the fungi species (Phytophthora spp.), while the rest, 10%, were attributed to orphan transcript‐derived fragments.

PCR: Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is one of the molecular techniques based on the modification of the polymerase chain reaction (PCR) method. RAPD makes use of a single, short, and arbitrary oligonucleotide primer that has the ability to anneal and prime at multiple locations throughout the whole DNA to produce a wide range of amplification products that are typically a characteristic of the template DNA that was used.

This means that in RAPD, segments of amplified DNA are random and the primers will bind somewhere in the sequence, though there is some uncertainty as to where exactly the binding will take place. For this reason, no prior knowledge of the DNA sequence for the targeted gene is needed.

As opposed to other normal PCR analyses, procedures that may involve the RAPD of the whole species (especially plant parts such as leaves) do not necessitate prior knowledge of the gene sequence of the organism under investigation. RAPD analysis is useful for the comparison of plant types. In this technique, decamer oligonucleotide primers (i.e. in a multiple of 10 bp each) are added to the DNA extracted from the species under investigation and then the whole mixture is subjected to PCR for amplification. The attractive features of RAPD include the fact that it is sensitive, fast, and does not make use of radioactive probes. However, RAPD has some limitations due to the fact that they are dominant alleles and therefore it is important to have many closely related markers to get reliable comparisons. Moreover, in cases where there is a mismatch between the primers and the templates, PCR products may not be observed, which may complicate the interpretation of the results. Another limitation comes from the fact that it is difficult to make a clear‐cut distinction between the amplified segments of the DNA and the heterozygous loci (a single copy) or homozygous (two copies), due to the fact that RAPD markers tend to be dominant. Moreover, with PCR being an enzymatic process, there is a possibility that the quality and quantity of both the template DNA, amount of PCR components, and the conditions of the PCR cycling, influence their products. This complicates the carrying out of the RAPD analyses, necessitating that the technique be done by qualified personnel in a well‐established laboratory.

RAPD Principle

Figure 11.1a,b summarizes the procedures (in two cycles), which can show the principle in which the technique operates:

Figure 11.1a Cycle one: Summary of the procedures (in two cycles), showing the principle in which the technique operates.

Figure 11.1b Cycle two: Summary of the procedures (in two cycles), showing the principle in which the technique operates.

Microsatellite Techniques

By definition, a microsatellite refers to segments of tandemly repetitive sequences of adjacent/allelic genes, for example (CA)n, which occurs at numerous eukaryotic genome locations. The general microsatellite procedures involve the PCR amplification of the region on the genome that contains the microsatellite using appropriate primers. The number of repeats on the allelic gene for the microsatellite under study will determine the size of the amplified DNA and the DNA fragments are then separated on a gel. There are several microsatellite‐PCR techniques that are used in food forensic studies and they include inter‐sequence simple repeat (ISSR)‐PCR and simple sequence repeats (SSR)‐PCR, etc.

Microsatellites sequences are also called by other names, such as short tandem repeats (STRs) or variable number of tandem repeats (VNTRs), as known by forensic geneticists who use the technique for forensic identification. Microsatellite is also known as simple sequence repeats (SSRs), mainly by plant geneticists who use the technique for DNA profiling, genetic linkage correlations, etc. Microsatellites occur mostly in gene introns (non‐coding regions of the DNA, thus biologically silent), do not have any consequence with regard to the functioning of the gene, but are used merely as markers to identify loci and some specific chromosomes. They are also not associated with causing disease by themselves. Microsatellites are well known for their diversities, for example in some of these microsatellites, the repeated unit (e.g. CA) may occur three times, in others it may occur ten times, etc., but generally more than twice. Microsatellites are also known for undergoing high rates of mutations and rely mostly on the variations in the number of repeats for the allelles being studied rather than on the number of repeats for the microsatellite under study.

In a population, if an organism’s DNA polymerase adds to the microsatellite, this will result in a larger copy of this repeated sequence, and will be passed on to subsequent generations for further replications, which will create the possibility for recombination over the repeated breeding cycles. This trend is meant to ensure that the variability of the repeated sequence in this particular population is maintained, making it characteristic for that population and distinct from all other populations that do not form part of the interbreeding.

There are a number of methods that are possible for use in the detection of microsatellites, but most studies have involved the use of polymerase chain reaction primers (PCR primers – Figure 11.2). These primers are designed such that they are highly specific and unique to one locus, such that just a single pair of PCR primers can be used for the identification of every individual in the same species, although they may result in products of varying sizes of the amplified products, depending on the different length microsatellites used.

Figure 11.2 Illustration of the PCR method used as a detection method for microsatellites.

Inter‐sequence Simple Repeat (ISSR)‐PCR

Inter‐sequence simple repeat (ISSR)‐PCR has several synonymous variants as follows: anchored simple sequence repeats (ASSR); anchored microsatellite primed (AMP)‐PCR; microsatellite primed (MP)‐PCR, which is an unanchored primer; simple sequence repeat (SSR)‐anchored PCR (Zietkiewicz et al., 1994); inter‐sequence simple repeat amplification; random amplified microsatellites (RAMs); random amplified microsatellite polymorphisms (RAMPs); and single primer amplification reaction (SPAR).

Genome Fingerprinting by Simple Sequence Repeat (SSR)‐anchored Polymerase Chain Reaction Amplification

The simple sequence repeat (SSR) technique combines the advantages of both AFLP and RADP and involves the use of the sequences of microsatellites as primer, together with PCR to create polymorphic markers for the loci present in the target genome (DNA) (Godwin et al., 1997). ISSR‐PCR does not require any construction sequence information/data, because the chain polymerase reaction procedures are included in the procedure itself. This attribute, together with the fact that low amounts of the DNA template are required and also the ISSRs are always randomly distributed over the whole genome, are the main advantages of ISSR‐PCR. However, the technique suffers from the fact that it may well be associated with phenomena related to non‐homology of DNA fragments with similar size, because the technique is multi‐locus. Another shortcoming is that ISSR has reproducibility problems, just as RAPD does.

However, despite these limitations, ISSR‐PCR is highly useful in forensic investigations of genetic identity, strain identity, etc.

Forensically Informative Nucleotide Sequencing (FINS)/DNA/PCR Sequencing/Barcoding

In the forensically informative nucleotide sequencing/barcoding (FINS)/DNA/PCR technique, highly conserved species specific mitochondrial genes, such as 12S rRNA, Cytochrome b, and 16S rRNA are amplified using universal primers for the identification of species, using the biological material presented as forensic evidence.

Species‐specific PCR Primers

Species specific primers are possible to design due to the availability of the information on the species genome sequences, also due to the possibility of identifying single base polymorphism. To be able to design such primers, one may have to employ stringent conditions to generate a fragment of the DNA that may then be visualized on agarose gel in the presence of the gene from a specific species. The advantage of this technique is that there is an assurance of getting the gene fragment amplified, unless there is a technical error. The limitations of this technique arise from the fact that prior knowledge of the specimen to be analyzed is essential and has to be available to enable the identification of the target species. Moreover, the technique requires the inclusion of controls to exclude the possibilities of false positive or false negative observations.

Multiplex PCR

Multiplex PCR is a variant of PCR‐specific primer techniques, which is capable of amplification of several targets of interest simultaneously, by using more than one pair of primers in one set of the reaction (Michelini et al., 2007).

Conclusions

Molecular biology‐based methods are very attractive in food forensic cases due to their specificity, as they target molecules such as nucleic acids, proteins which are specific for each individual organism. These methods provide more reliable evidence. However, they require highly skilled personnel and advanced equipment, expensive reagents, and a laboratory environment that is clean and sterile.

A combination with other analytical/bioanalytical methods for a concrete proof of the results may be mandatory.

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