Diverse ecosystems, diverse species of plants and so are the techniques to study them. DNA fingerprinting of plants involve differentiating these diverse species using different genetic DNA based markers.
Genetic markers are any assayable trait for which alleles at the individual loci are inherited as Mendelian traits. Genetic markers are either morphological, biochemical or molecular. Conventionally, morphological markers have been used. Though simple and irreplaceable, their expression may be influenced by the environment leading to errors. Biochemical techniques detect variation at the gene level e.g. allozymes. Their utility is hindered by the relatively small number of loci that can be assayed in any population and also by technical complexity of their use. Furthermore, it has problems related to the specificity and age of the tissue. These limitations led to the use of molecular markers. Large numbers of DNA based markers are available and these have many important applications including fingerprinting and identification of germplasm, inferring phylogenetic relationships between species etc.
Different types of techniques can be distinguished based upon the main methods of analysis e.g. RFLP, VNTR based on southern hybridization, RAPD based on PCR or techniques generating sequence information such as SNP.
Every story however starts from only one point, DNA extraction. Many protocols have been proposed which will depend on the sample, quantity and quality of DNA needed, and substances interfering. The extracted DNA is then treated and analyzed in different ways according to the technique used.
Hybridization based methods:
RFLP, Restriction fragment length polymorphism markers, based on DNA-DNA hybridization were developed in 1970s. It differentiates on the basis of different patterns obtained after digesting with restriction enzymes. Restriction endonucleases usually HindIII, EcoRI, BamHI are used to cleave the DNA into fragments of varying length and sequences. These fragments are then separated by agarose gel electrophoresis and later transferred to nylon membrane using southern blotting. The fragments are then detected by hybridizing with labelled probes.
RFLP markers have many advantages as reproducibility, codominant inheritance, no sequence information requirement and easy scoring. But it has certain limitations also like it requires large amount of DNA, making probes is also difficult due to the presence of the large amount of repetitive DNA in nuclear genome and therefore its analysis is also problematic. The technique was also time consuming and expensive.
PCR based methods:
Kary Mullis, with the development of PCR revolutionized every field of molecular biology. Different techniques were developed using PCR which didn't have limitations of RFLP.
1. Random amplified polymorphic DNA (RAPD) markers are the simplest ones as no sequence information is required. It is based on the use of arbitrary primer generally of 10 base pairs and random sequence. It has to be taken care primer has more than 40% GC content and absence of the palindromic sequences. The primer binds to different loci on the DNA template and amplifies random fragments which help in distinguishing between different species. PCR product is run on agarose gel and analyzed by scoring the presence or absence of polymorphic bands.
Main advantage of these PCR dependent techniques is requirement of less amount of DNA template (around 10 ng per reaction). Also it is simple, costs less and does not require any prior sequence information. But many limitations are also there like lack of reproducibility as it depends on PCR conditions, quality of DNA template etc. Also RAPD markers are dominant markers i.e. heterozygous or homozygous loci can't be distinguished.
2. Amplified fragment length polymorphism (AFLP) markers combines RFLP and PCR both. DNA is first digested with restriction enzymes, ligated to adapters and then PCR is performed.
They are really good for fingerprinting as they give large number of polymorphic bands per gel and they are more repeatable than RAPD. But it also has limitations of being dominant, technically demanding and requiring more template DNA.
Sequence based markers:
Many repetitive sequences are present in the genomes of higher organisms, some of which can serve as genetic markers. Most polymorphic repetitive sequences are minisatellites and simple-sequence repeats (SSRs or microsatellites).
Minisatellites consist of regions containing repeat sequences 16 to 64 base pairs. Different individuals have different number of repeats as mutation rate is high in these regions. Thus these markers show high level of polymorphism and are reproducible also.
2. Simple sequence repeats (SSRs):
Microsatellites or SSRs have tandem repeats of 1-6 base pairs. The technique is having number of advantages as it is reproducible, shows codominance, highly polymorphic for even closely related species, can be easily automated and requires only 10-100 ng of template DNA.
The main disadvantages are difficulty in making genomic libraries rich in microsatellites and designing of primers for which sequence of the genome has to be known.
3. Inter-simple sequence repeat (ISSR) :
It involves amplification of region between microsatellites as they are used as primers itself. Many times primers are anchored on 3' or 5' end with selective nucleotides. Thus, the technique does not require prior sequence information still showing the specificity as SSRs. Moreover, it is reproducible, fast and simple.
4. Expressed Sequence Tags (ESTs):
These are short DNA molecules, 5' or 3' flanking region of cDNA as they are formed by reverse transcription of mRNA. There are large numbers of ESTs present in computerized database now and these are even used to prepare other molecular markers.
5. Single Nucleotide Polymorphism (SNPs):
SNP is the polymorphism between two genomes based on a single nucleotide difference. These may be analyzed by different methods such as gel electrophoresis, fluorescence resonance energy transfer (FRET), luminescence, mass spectrophotometry (MALDI-TOF), chromatography etc.
6. Sequence characterized amplified region (SCAR):
The two ends of RAPD markers which have been important in some diagnosis are sequenced and cloned to form primers for SCAR. Thus they detect only single locus and are less dependent on the conditions of the reaction.
With time and new requirements, many molecular markers are being developed. The desirable properties are reproducibility, codominance, easy to use, cost effective, and anticipated polymorphism. All properties cannot be found in any one of the marker. The choice of the marker to be used thus depends on the researcher only and the project as each molecular marker has at least some of the desirable properties.
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