DNA Fingerprinting and RAPD
“DNA Fingerprinting — Greatest breakthrough in forensic sciences in this century”
Identification and characterization of individuals is carried out at different levels. These can be social, physical or biological. The biological identity means phenotypic and genotypic markers. Most commonly used biological markers for individual identification include blood groups, serum proteins, enzymes, etc. These markers have proved useful but they are limited in number and their degree of variation. So these cannot be used in precise identification of specific individual. Most of the genomes of animals and plants cannot vary greatly between individuals because it has an essential coding function. In non-coding regions this requirement does not exist and the DNA sequence can accommodate changes. One change, which does occur, is the tandem repetition of DNA sequences. The discovery of hypervariable repeats (HVR) in human DNA has created a powerful new class of genetic markers, which promise to revolutionize forensic biology, and opened new vistas in animal and plant sciences. The HVR, also referred to as mini-satellites or variable number of tandem repeats (VNTRs), consists of core tandem repeats of a short nucleotide sequence about 15-30 base pairs in length. They are hyper-variable because the number of tandem repeats, and hence the length of DNA in that region, varies considerably in the general population. DNA probes have been isolated which detect families of these HVR located at many different chromosomal loci. The probability that two unrelated individuals have identical lengths of DNA at a particular HVR is very low. However, the probes that have been developed to detect 30-40 different HVR simultaneously, so the probability that all of these are the same length in both individuals becomes vanishingly small. The complex banding pattern obtained when southern blots of DNA are hybridized with these probes is therefore individual specific, and is referred to as a DNA fingerprint.
DNA identification analysis, identity testing, profiling, finger-printing, typing or genotyping all refer to the same phenomenon of characterization of one or more rare features of an individual’s genome or hereditary make up by developing DNA fragment band (alleles) patterns. If sufficient number of different size bands is analyzed, the resultant bar code profile will be unique for each individual except identical twins. The bands of DNA profile are inherited in a simple Mendelian fashion and behave as co-dominants; the maternal and paternal derived variants at any given locus are detectable. DNA fingerprinting is considered most important among various genome markers and like restriction enzyme length polymorphism there being genetic loci at which nearly every individual is unique and different. In such a case an offspring would inherit one or the other of the allelic status of each marker from each parent. Another offspring of the same parents would again inherit markers from the parents, but it would be different set. Thus, some markers in the two off-springs would be same and others would be different. On the other hand, two unrelated individuals would possess virtually no markers in common. Proponents of DNA fingerprinting claim that the probability of two DNA samples matching by chance is very low, somewhere between 10-6 to 10-15.
The principle of individual uniqueness and identical DNA structure within all tissues of the same body provides the basis for DNA fingerprinting. Jeffreys and his colleagues developed DNA fingerprint system utilizing the nature of hyper-variability of these regions in human beings. The advent of DNA fingerprint technique has revolutionized the identification of any biological specimen by dramatically reducing the number of tests required, yet radically increasing the power of identification precisely. Impact of DNA fingerprinting technique on science, law and politics has been dramatic. Recently this technology has been found to have many applications in livestock, primates, birds and other species.
Genetic Basis of DNA Fingerprinting
The four bases of DNA are organized in different ways in DNA sequence, which is quite variable in individuals. The repetitive sequences generally comprise 2-250 bp of a specific sequence, which is typically repeated between a few to several thousand times. The resulting multiplicity of lengths of these segments is called length polymorphism (HVR or VNTR). The development of different probes provided tools for the observation of numerous hybridization signals and thus a large number of bands (DNA fingerprinting). Using appropriate stringency hybridization conditions, highly polymorphic DNA bands scattered throughout the entire genome of humans and animals can be detected.
The methods used in obtaining DNA fingerprinting are conventional techniques of molecular biology. Each technique has many alternative protocols; however, one should standardize protocols suitable to the conditions and facilities available in the laboratory. The general outlines of the procedure is as follows:
- Isolation of DNA- DNA can be isolated from any tissues containing nucleated cells. However, in animals quite commonly used sources of DNA are blood, semen, etc. Leukocytes or sperms from these tissues are lysed using specific buffers and subjected to proteinase-K digestion. Digested proteins are precipitated and DNA is removed with the help of repeated phenol- chloroform-isoamly alcohol extraction technique. DNA is finally precipitated using isopropyl alcohol. Subsequently, its quality and quantity should be checked.
- Digestion of DNA by restriction endonuclease – About 5-10 g DNA is sufficient to get good quality fingerprinting. Commonly used enzymes are EcoR1, Hae III, Alu 1, etc. A typical reaction mixture for DNA digestion contains DNA, enzyme, buffer specific for each enzyme, BSA, distilled water. The reaction mixture is incubated overnight at a specific temperature. The digestion is stopped by heating or addition of EDTA.
- Electrophoretic separation of different fragments — The digested DNA is an admixture of DNA fragments of various sizes. These segments are separated according to their sizes by electrophoresis. Appropriate DNA size markers are also used on the side lanes of DNA samples.
- Transfer of DNA on nylon membrane – The size separated DNA fragments should be transferred to a solid surface usually a nitrocellulose or nylon membrane for further use by adopting a method known as southern transfer. Transfer can be accomplished by using either capillary or vacuum transfer method.
- Probe labeling – A DNA probe is a stretch of DNA fragment, which is complementary to target sequences in the genome. In DNA fingerprinting the probe will be complementary to repeating units of DNA or also known as satellite DNA which falls under different categories depending on size of the repeating unit. The probes are labeled with 32P radioactive labelled nucleotides.
- Hybridization— The labeled probe DNA should be hybridized with the complementary sequences located on the nylon membrane for the detection of position of the latter. It involves incubation of nylon membrane with the labeled probe in proper hybridization solution at appropriate temperature.
- Autoradiography — It involves alignment of hybridized nylon membrane with a X-ray film in a cassette followed by a specific duration of incubation at a low temperature. The X-ray film alter its development shows lanes with bands or multiple number of bands that look like bar codes otherwise known as DNA fingerprints.
- Analysis and interpretation of band patterns — It is done by comparison of position of bands and by band sharing tendency using various computer softwares.
Different areas of animal science where DNA fingerprinting has a great potential are:
- Individual identification
- Pedigree analysis and parentage verification
- Conservation of genetic resourced
- Zygosity testing
- Demographic studies
- Quality control of cell banks
- Sex determination
- Detection of loci controlling quantitative traits or disease resistance
- Pathogen identification
- Identification of carcass of tissues
- Detection of somatic mutations of cancer
- Taxonomic tool
RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)
Advances in molecular biology techniques have provided the basis for unraveling virtually unlimited numbers of DNA markers. The utility of DNA-based markers is generally determined by the technology that is used to reveal DNA-based polymorphism. Presently, the restriction fragment length polymorphism (RFLP) assay has been the choice for many species to measure genetic diversity and construct a genetic linkage map. However, an RFLP assay which detects DNA polymorphism through restriction enzyme digestion, coupled with DNA hybridization, is, in general, time consuming and laborious. Over the last decade, polymerase chain reaction (PCR) technology has become a widespread research technique and has led to the development of several novel genetic assays based on selective amplification of DNA. This popularity of PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately, because of the need for DNA sequence information, PCR assays are limited in their application. The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome facilitated the development of genetic markers for a variety of purposes. The simplicity and applicability of the RAPD technique have captivated many scientist interests. Perhaps the main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterization of the genome of the species to be analyzed. Therefore, Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments obtained by PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.
RAPD technique uses short synthetic oligonucleotides (approximately 10 bases long) of random sequences as primers to amplify small amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are then separated on agarose gels and stained with ethidium bromide. Welsh and McClelland independently developed a similar technique using primers approximately 15 nucleotides long and different amplification and electrophoretic conditions from RAPD and called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique. PCR amplification with primers shorter than 10 nucleotides [DNA amplification fingerprinting (DAF)] has also been used producing more complex DNA fingerprinting profiles. At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other. The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNA will result in the presence or absence of bands because of changes in the priming sites. The profile of RAPD bands is similar to that of low stringency mini-satellite DNA fingerprinting patterns and is therefore also termed RAPD fingerprinting. RAPD are dominant markers.
RAPD is a simple and cost effective technique because of which it has found a wide range of applications in many areas of biology. Some of the areas where the technique is used are as follows:
- Genetic Mapping— Restriction fragment length polymorphisms (RFLPs) have been commonly used to map genes. This approach involves hybridization of a probe to Southern blotted genomic DNA digested with restriction endonucleases. A useful probe will detect differences in restriction fragment lengths arising from loss or gain of recognition sites or from deletions or insertions of stretches of DNA between sites. The speed and efficiency of RAPD analysis encouraged scientists to perform high-density genetic mapping in many plant species such as alfalfa, faba bean and apple in a relatively short time.
- Developing Genetic Markers Linked to a Trait— One of the most widely used applications of the RAPD technique is the identification of markers linked to traits of interest without the necessity for mapping the entire genome.
- Population and Evolutionary Genetics—The RAPD technique has received a great deal of attention from population geneticists because of its simplicity and rapidity in revealing DNA-level genetic variation, and therefore has been praised as the DNA equivalent of allozyme electrophoresis. A major drawback of RAPD markers in population genetic studies of outbreeding organisms is that they are dominant. Thus gene frequency estimates for such loci are necessarily less accurate than those obtained with co-dominant markers such as allozymes and RFLPs.
- Reproducibility of RAPD Markers—RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. Perhaps some primers do not perfectly match the priming sequence, amplification in some cycles might not occur, and therefore bands remain fainter. The chance of these kinds of bands being sensitive to reaction conditions of course would be higher than those with higher intensity amplified with primers perfectly matching the priming sites. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA.
- Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co- dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.
- PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is laboratory dependent and needs carefully developed laboratory protocols to be reproducible.
- Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.
RAPD markers have found a wide range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant, and animal breeding. This is mainly due to the speed, cost, and efficiency of the RAPD technique to generate large numbers of markers in a short period compared with previous methods. Therefore, RAPD technique can be performed in a moderate laboratory for most of its applications. Despite the reproducibility problem, the RAPD method will probably be important as long as other DNA-based techniques remain unavailable in terms of cost, time and labor.