Techniques - DNA

Genomic vs Mytochondrial DNA

DNA can be located in the cell nucleus (genomic DNA) or in cell organelles (mitochondria in animals/plants, or also chloroplasts in plants). Genomic DNA has more differential coding regions between closely related species but is present at relatively low copy numbers. Mitochondrial DNA is less discriminatory but is present at much higher copy numbers.

DNA Extraction

Almost all analyses require the extraction of DNA from the sample. The DNA may be intact, or may be fragmented (e.g. in a processed food). Extraction conditions may need to be optimised for each sample type. There are a number of proprietary extraction reagents and systems, with Qiagen having dominated the market in recent years. A significant proportion of a DNA assay cost can be the licencing cost of the extraction reagents.

DNA Amplification (PCR)

As the quantity of DNA from the sample is very small, and most DNA assays are only analysing relatively small target fragments of the DNA, then amplification is required for most applications.

Amplification is in 3 steps:

1. denaturation, in which double-stranded DNA templates are heated to separate the strands.
2. annealing, in which the primers bind to flanking regions of the target DNA.
3. elongation (extension), in which DNA polymerase extends the 3′ end of each primer along the template strands with the bases.

These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA.

Image by Enzoklop, Wikimedia, licensed under Creative Common Attribution

RT-PCR (Real-Time PCR) or qPCR (Quantitative PCR)

RT-PCR is an adaption of PCR, whereby the progress of amplification of the target DNA can be followed as it is occurring.  It enables a quantitative determination of the amount of target DNA in the sample. It is achieved by combining a fluorescent dye and quencher into the primers.  At the start of the process there is no fluorescence. As the PCR reaction proceeds, the target DNA is amplified, and the quencher and fluorescent dye is released every time a cycle and polymerisation are completed. The level of fluorescence is read against the number of cycles. Monitoring the fluorescence signal vs cycle numbers gives a characteristic flat baseline, followed by steep increase, followed by plateau .

 Image by Genetic Education Inc, licensed under Creative Common Attribution

The steep increase corresponds to the point where DNA copy numbers begin to rise exponentially from a baseline. The number of cycles before this steep curve occurs is indicative of the number of DNA copies in the original mix and is calculated as the threshold value.

The RT-PCR is run not only with the target DNA from the sample gene, but also a similar target sequence from a housekeeping gene (or standard). The DCt value is then the difference between the sample C_t value and the housekeeping gene C_t value.

RT-PCR  can be set up as a multiplex assay with many different primers in the same mix e.g., where many species of meat can be analysed together

Digital PCR (dPCR)

Digital PCR is based on the fragmentation of the PCR system into extremely small and numerous compartments: using a statistical model (Poisson).  The aim is to create conditions where each DNA molecule of the mix becomes trapped in a mini reactor. Using fluorescence, each reactor showing a signal therefore equates to one DNA molecule.

Droplet Digital PCR (ddPCR) technology is a further refinement. Droplets are formed in a water-oil emulsion system to form the partitions that separate the template DNA molecules. The droplets serve the same function as individual test tubes or wells in a standard dPCR plate, although in a much smaller format. The reagents are the same as a normal PCR reaction, and PCR amplification of the template DNA occurs in each individual droplet. No standard curve is required, and the fluorescence produced by the PCR is measured at the end of the all the PCR cycles to give copies per microlitre.

In comparison to RT-PCR, dPCR measures the actual number of molecules (target DNA). It provides absolute quantification because dPCR measures the positive fraction of samples, which is the number of droplets that are fluorescing due to amplification. This positive fraction accurately indicates the initial amount of template nucleic acid. RT-PCR on the other hand measures the intensity of fluorescence at specific times (generally after every amplification cycle) to determine the relative amount of target molecule (DNA). It cannot specify the exact amount without constructing a standard curve using different amounts of a defined standard. It gives the threshold per cycle (CT) and the difference in CT is used to calculate the amount of initial nucleic acid. As such, RT-PCR is an analogue measurement, which may not be as precise due to the extrapolation required to attain a measurement.

DNA assays are principally used for determining species of animals or varieties of plants, and RT-PCR in particular can give quantitative results. Where specific varieties of plant foods or breeds of animals are linked to specific regions or countries, then DNA analysis can confirm geographical origin as well. Applications developed within the UK Defra Food Authenticity programme include species identification in commercial meat species, exotic meat species, bushmeat, fish and seafood, undeclared meat ingredients in vegetarian foods, potato varieties, fruit varieties, Basmati rice,  durum wheat pasta,  GMO ingredients in foods,  hazelnut oil in olive oil, mandarin in orange juice.

The wide variety of analytical configurations is driven by the variety of ways to analyse and interpret the amplified DNA.  Some of the most common are given below.

Restriction Fragment Length Polymorphism – RFLP

This method cuts the amplified target DNA into smaller fragments using an enzyme. The enzyme cuts at specific base pairs, hence the distribution of the fragment lengths will be specific to the species or plant varieties being sampled. This assay is one of the simplest methods to detect and identify SNPs (single nucleotide polymorphisms), where a species has only one base differences to the main family gene sequence. The different length fragments can then be separated by (capillary) chromatography, giving a chromatogram where the retention time is indicative of the fragment length and the peak area is indicative of the fragment concentration (and hence the copy numbers in the sample).

An example of the approach was the development of identification of fish species using the Agilent lab-on-a-chip capillary electrophoresis system. Because families of fish are quite closely related, the fragment distribution of 3 restriction enzymes were used in turn, The distribution can then be part of a database, which permits unknown species samples to be identified by comparing the fragments distribution with the database.

Amplified Fragment Length Polymorphism - AFLP

This is sometimes referred to as AFLP-PCR, as it has a further PCR step after the first amplified target DNA has been cut with an enzyme. Adapters are then linked to the end of the DNA fragments. A subset of the fragments is then selected to be amplified. This selection is achieved by using primers complementary to the adaptor sequence, the cutting site sequence and a few nucleotides inside the resultant fragments. The amplified fragments are then separated by (capillary) electrophoresis to give a fragment profile fingerprint which again serves as database of authentic species/varieties.

AFLP is mainly used for differentiating very closely related species of plants, fungi, animals, and bacteria and other microorganisms.

DNA Sequencing - Barcoding

This method is mainly used for species or varietal identification. The PCR reaction amplifies a targeted generic gene sequence which occurs in most animal species or one which occurs in most plants .

The target gene sections are then sequenced by selectively including polymerase inhibitors into the PCR mix.  This is known as Sanger sequencing.  Bases can then be separated by capillary electrophoresis and compared with bioinformatic databases (usually prepared from full genome sequencing) to identify the species or varieties.

DNA Sequencing - NGS – Next Generation Sequencing

This technology has begun to revolutionise DNA methodology and has to a large extent replaced RFLP and AFLP approaches. Like barcoding, universal primers are used to amplify a short but variable genomic section present in all biological species. The amplified product is subject to a rapid sequencing technology (NGS), which independently sequences each strand of DNA from the PCR reaction. The output of this sequencing is a data file of thousands to millions of DNA sequences, which are then compared to a bioinformatic reference database to identify all the species/varieties in the sample.

NGS is particularly useful when there are mixed species/varieties to identify as it is a rapid method giving results quite quickly compared to Sanger sequencing.

DNA Hybridisation Probes and Melting Curves

DNA can also be selectively detected by the use of hybridization probes. These are sequences of bases that have been designed to form complementary strands with specific regions of target DNA in the sample. The probes are labelled to enable easy detection, for example with an antigen, a radiolabel, or a fluorescent tag. One common way of using the technique is to design a fluorescent tag that is quenched when the probe binds with its target. The mix is then slowly heated, and at a certain temperature the bound strands will separate, and the probe fluoresces again. The plot of fluorescence against temperature (the “melting curve”) is characteristic of the strength of binding; it will be strongest when the bound DNA is an exact match for the probe (i.e. when the sample contains the target DNA).

Single Sequence Repeats (SSRs) or Microsatellites or Short Tandem Repeats (STRs)

Image by the authors

 This approach is particularly used for identification of plant varieties. It uses the DNA sequences that occur in the non-coding regions between the genes. These have repeat base sequences often off three or four bases. In Figure 11, the repeat sequence is CGG, and plant 1 has 5 repeats, plant 2 has 7 repeats, plant 3 has 3 repeats, and plant 4 has 5 repeats. These are amplified by PCR using the appropriate primers, and then separated by electrophoresis. The gel or capillary gives either bands or a chromatogram of alleles. By using a set of different primers, a profile of different alleles can be built up for the different plants and used to identify an unknown variety.

This assay was used for many years to identify Basmati and non-Basmati varieties of rice using a 10-marker primer assay. This was a very effective method based on the unique organoleptic properties of Basmati, until a lot more varieties of Basmati were approved, in which case this approach was not able to distinguish all the new varieties.

KASP (Kompetative Assay Specific PCR)

KASP is used to detect single nucleotide polymorphisms (SNPs) (i.e. where there is only one base difference in the target sequence) or inserts and deletions (INDELS). It completes a double PCR reaction, where the forward primers is designed to bind solely with the SNP, and the reverse primer produces a complementary strand on the opposite strand of the DNA. In the second PCR reaction, the complementary strand to the allele-specific forward primer is generated when the common reverse primer binds to the amplicon formed in the first round of PCR. The primer is fluorescently labelled and designed to released fluorescence as the second PCR reaction proceeds. The assay is therefore verifying the specific SNP that occurs in the sample.

Image by JP Livingstone, Wikimedia, licensed under Creative Common Attribution

 In order to develop a varietal verification, an assay using 8 or so KASP primers is used. A KASP method has been developed to identify all the new 25 varieties of Basmati rice.

 LAMP (real time fluorescence loop-mediated isothermal amplification) Assay

This is a technique that avoids thermocycling PCR. It is a novel nucleic acid amplification method that is performed under isothermal conditions utilising the Bst from Geobacillus stearothermophilus DNA polymerase. The target sequence is amplified at a constant temperature of 60–65 °C using either two or three sets of primers and can be carried out even in a ‘thermos flask’. The Bst polymerase has a high strand displacement activity in addition to a replication activity. 

The assay was originally developed to identify different viruses but has been adapted for many food authenticity applications, including authenticating Atlantic salmon by combining a fluorescent dye with primers which is released during the amplification.

CRISPR/Cas9 Assay

CRISPR/Cas9 is a gene editing system, which can be applied to authenticate food. Cas9 is an enzyme which cuts the DNA in a specific place, and permits editing to take place. When used in an assay, the fragments produced after cutting are identified, and used as markers to authenticate the food.

In one early food authentication application, researchers  developed a new CRISPR/Cas9 based in vitro assay to distinguish a fine cocoa variety from a bulk cocoa variety. The bulk cocoa contains a SNP which is located within a PAM region (protospacer adjacent motif) mandatory for the Cas9 endonuclease. Consequently, only the bulk cocoa is attacked by the enzyme. The result can be recorded using agarose or capillary gel electrophoresis (AGE and CGE).

Provided the validation of assays has been properly conducted as to whether there is any cross-reactivity between the designed primers with other species or varieties, then there is a very high level of confidence with the identification. DNA assays and RT-PCR can be multiplexed to be able to analyse for many species/varieties in one action of the assay. Although originally sequencing was expensive and slow, the latest development of NGS has revolutionised sequencing of sections of genes. Also, the cost and throughput time of WGS (whole genome sequencing) has been considerable reduced.

Although in general DNA assays require skilled laboratory technicians, recent developments have also made it possible to undertake on-site DNA assays.

When designing primers for PCR reaction, it is important that the fragment length is appropriate for the sample i.e. if the sample has been processed and the DNA has been fragmented, the fragment length should be relatively short (less than 200 base pairs) or the PCR reaction will not work. Quantitative determinations are measuring copy numbers, and this has to be related back to concentration (e.g., g/100g), which can be difficult. The amount of original DNA can vary from animal to animal, cut to cut or maturity of plants, and it is important that genome DNA is used and not mitochondrial DNA.

RT-PCR

RT-PCR is measuring copy numbers of the DNA rather than the concentration (e.g. weight/weight % composition) of the test species. To try and convert to concentration, a standard curve is produced of DCt values against different concentrations of the species/varieties. This enables the DCt value of an unknown sample

e.g. Standard curve of T. aestivum (common wheat)  linked to its concentration in the durum wheat sample (Crown copyright image)

However, as DNA copy numbers will vary from cut to cut of meat, fish and even plant foods in the raw material or processed composite product, the value of w/w obtained may not be so accurate. It is also important that RT-PCR determinations do not use mitochondrial DNA, which has multi-copies of DNA, but genomic DNA.