DNA fingerprinting also termed as DNA profiling, is a method of isolating and identifying variable elements within the base pair sequence of DNA. It is useful in identifying individuals particularly in forensic sciences. It is helpful in establishing a link between biological evidence and a suspect.
Technique for DNA fingerprinting:
It involves a process termed as Southern blotting. The credit goes to E.M. Southern for its discovery. A very small sample of DNA is sufficient for the test. The sample includes blood, semen, urine, hair follicles, etc.
Following are some of the major steps of DNA fingerprinting technique :
- The first step is the separation of DNA from blood, semen, hair follicles, etc.
- The amount of DNA obtained from only a few cells is generally small. Polymerase chain reaction creates a large number of copies of this DNA.
- The DNA sample is cut into small pieces with the help of suitable restriction enzymes.
- The complex mixture of fragments is subjected to gel electrophoresis. The process requires agarose polymer gel. The next step is the staining of DNA fragments with the help of fluorescent dye.
- The restriction (DNA) fragments present in the gel are denatured (split) with alkali.
- Denatured DNA sequences are transferred on to a nitrocellulose filter on nylon membrane. The procedure is termed as ‘Southern Blotting’. The process preserves the distribution of fragments in the gel, while creating a replica on the filter.
- The filter is then incubated with specific radioactive probes or markers. The DNA sequences complementary to VNTR (or fragments in the gel) acquire radioactivity due to hybridization with probes.
- The probe bound to the filter or sheet is detected by autoradiography, i.e., by exposing it to the x-ray film. DNA fragments appear as dark bands at the probe sites.
All DNA testing is based on the observation that the genome of each person or animal is unique (except identical twins). DNA polymorphism is the result of mutations or variations. Mutations can occur in the somatic (vegetative) as well as in reproductive (germ) cells. The somatic mutations do not transmit from parents to the offspring. The germinal mutations those occurring in reproductive cells often transfer to the offsprings through the gametes. Individuals of future generations thus produced carry these mutations.
Such mutations or variations in DNA sequences of alleles occur very commonly. When these mutations occur in more than one allele at a locus with a frequency of more than 0.01% in a population, then it is termed as DNA polymorphism . In other words, if a heritable mutation in a population shows higher frequency it is termed as DNA polymorphism.
The differences in nucleotide sequence among individuals are DNA polymorphisms.
Mutations could occur either in coding sequences or non coding sequences DNA. While mutations in coding sequences affect the organism, those in non-coding sequences have no immediate effect. Therefore, non coding sequences continue to accumulate for several generations. This produces large variability in human population. The variability ranges from single nucleotide change to changes in large number of variations. In fact these changes form the very basis of evolution.
Two fundamentally different types of polymorphism useful for DNA typing are tandem repeats and restriction fragment length polymorphisms.
Tandemly repeated DNA
The eukaryotic genome has large number of short sequences that repeats over and over in small to large arrays termed as minisatellites and microsatellites. Another term commonly used to describe these sequences is variable number tandem repeats or VNTRs.
For a given locus, the number of repeats is highly variable among individuals and heterozygosity is high (i.e., the number of repeats at the locus is usually different on the two pairs of chromosomes of one individual). Analyzing the number of repeats at one or more such loci provides a highly sensitive measure of individual identity.
Variability in restriction sites
Single base changes in DNA often introduce or obliterate a restriction enzyme site. For example, a mutation that changes the sequence AGATCC to GGATCC will introduce a Bam H1 site into that segment of DNA. Such sequence variability is exceedingly common particularly in non coding regions of the DNA and determining whether or not a particular group of restriction sites exists in DNA is a very sensitive means of differentiation one individual from many others.
Because polymorphism in a restriction site translates into variability in the length of fragments after digestion of DNA with restriction enzyme, these DNA markers are termed as restriction fragment length polymorphism or RELPs.
Application of DNA fingerprinting
DNA fingerprinting as evidence has already been legally accepted. Following are some of the applications of DNA fingerprinting:
- DNA sequencing (fingerprinting) helps in determining the paternity of a child. The claims of being a father or mother of child can be verified.
- The identity of a criminal, like rapist, murderer,etc., can be established. The only material needed is a small DNA sample from blood stains, semen, urine, hairs, saliva, perspiration,etc., even if these are several years old.
- It can also be used to determine whether two persons are closely related.
- DNA sequences can also unravel the course of biological evolution by establishing relationship between different races.
- DNA in analysis also helps in diagnosis of inherited human diseases.
- By matching DNA of different persons, their relationships can be ascertained.
- By matching DNA samples different racial groups, their relationships can be found out. It will help in understanding biological evolution.