As stated in the preceding section, 99.9 per cent of base sequence among humans is the same. Assuming human genome as 3 × 109 bp, in how many base sequences would there be differences? It is these differences in sequence of DNA which make every individual unique in their phenotypic appearance. If one aims to find out genetic differences between two individuals or among individuals of a population, sequencing the DNA every time would be a daunting and expensive task. Imagine trying to compare two sets of 3 × 106 base pairs. DNA fingerprinting is a very quick way to compare the DNA sequences of any two individuals.
DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA, because in these sequences, a small stretch of DNA is repeated many times. These repetitive DNA are separated from bulk genomic DNA as different peaks during density gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. Depending on base composition (A : T rich or G:C rich), length of segment, and number of repetitive units, the satellite DNA is classified into many categories, such as micro-satellites, mini-satellites etc. These sequences normally do not code for any proteins, but they form a large portion of human genome. These sequence show high degree of polymorphism and form the basis of DNA fingerprinting. Since DNA from every tissue (such as blood, hair-follicle, skin, bone, saliva, sperm etc.), from an individual show the same degree of polymorphism, they become very useful identification tool in forensic applications. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes.
As polymorphism in DNA sequence is the basis of genetic mapping of human genome as well as of DNA fingerprinting, it is essential that we understand what DNA polymorphism means in simple terms. Polymorphism (variation at genetic level) arises due to mutations. (Recall different kind of mutations and their effects that you have already studied in Chapter 5, and in the preceding sections in this chapter.) New mutations may arise in an individual either in somatic cells or in the germ cells (cells that generate gametes in sexually reproducing organisms). If a germ cell mutation does not seriously impair individual’s ability to have offspring who can transmit the mutation, it can spread to the other members of population (through sexual reproduction). Allelic (again recall the definition of alleles from Chapter 5) sequence variation has traditionally been described as a DNA polymorphism if more than one variant (allele) at a locus occurs in human population with a frequency greater than 0.01. In simple terms, if an inheritable mutation is observed in a population at high frequency, it is referred to as DNA polymorphism. The probability of such variation to be observed in non-coding DNA sequence would be higher as mutations in these sequences may not have any immediate effect/impact in an individual’s reproductive ability. These mutations keep on accumulating generation after generation, and form one of the basis of variability/polymorphism. There is a variety of different types of polymorphisms ranging from single nucleotide change to very large scale changes. For evolution and speciation, such polymorphisms play very important role, and you will study these in details at higher classes.
The technique of DNA Fingerprinting was initially developed by Alec Jeffreys. He used a satellite DNA as probe that shows very high degree of polymorphism. It was called as Variable Number of Tandem Repeats (VNTR). The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as a probe. It included
(i) isolation of DNA,
(ii) digestion of DNA by restriction endonucleases,
(iii) separation of DNA fragments by electrophoresis,
(iv) transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon,
(v) hybridisation using labelled VNTR probe, and
(vi) detection of hybridised DNA fragments by autoradiography. A schematic representation of DNA fingerprinting is shown in Figure 6.16.
Figure 6.16 Schematic representation of DNA fingerprinting: Few representative chromosomes have been shown to contain different copy number of VNTR. For the sake of understanding different colour schemes have been used to trace the origin of each band in the gel. The two alleles (paternal and maternal) of a chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime scene matches with individual B, and not with A.
The VNTR belongs to a class of satellite DNA referred to as mini-satellite. A small DNA sequence is arranged tandemly in many copy numbers. The copy number varies from chromosome to chromosome in an individual. The numbers of repeat show very high degree of polymorphism. As a result the size of VNTR varies in size from 0.1 to 20 kb. Consequently, after hybridisation with VNTR probe, the autoradiogram gives many bands of differing sizes. These bands give a characteristic pattern for an individual DNA (Figure 6.16). It differs from individual to individual in a population except in the case of monozygotic (identical) twins. The sensitivity of the technique has been increased by use of polymerase chain reaction (PCR–you will study about it in Chapter 11). Consequently, DNA from a single cell is enough to perform DNA fingerprinting analysis. In addition to application in forensic science, it has much wider application, such as in determining population and genetic diversities. Currently, many different probes are used to generate DNA fingerprints.
SUMMARY
Nucleic acids are long polymers of nucleotides. While DNA stores genetic information, RNA mostly helps in transfer and expression of information. Though DNA and RNA both function as genetic material, but DNA being chemically and structurally more stable is a better genetic material. However, RNA is the first to evolve and DNA was derived from RNA. The hallmark of the double stranded helical structure of DNA is the hydrogen bonding between the bases from opposite strands. The rule is that Adenine pairs with Thymine through two H-bonds, and Guanine with Cytosine through three H-bonds. This makes one strand complementary to the other. The DNA replicates semiconservatively, the process is guided by the complementary H-bonding. A segment of DNA that codes for RNA may in a simplistic term can be referred as gene. During transcription also, one of the strands of DNA acts a template to direct the synthesis of complementary RNA. In bacteria, the transcribed mRNA is functional, hence can directly be translated. In eukaryotes, the gene is split. The coding sequences, exons, are interrupted by non-coding sequences, introns. Introns are removed and exons are joined to produce functional RNA by splicing. The messenger RNA contains the base sequences that are read in a combination of three (to make triplet genetic code) to code for an amino acid. The genetic code is read again on the principle of complementarity by tRNA that acts as an adapter molecule. There are specific tRNAs for every amino acid. The tRNA binds to specific amino acid at one end and pairs through H-bonding with codes on mRNA through its anticodons. The site of translation (protein synthesis) is ribosomes, which bind to mRNA and provide platform for joining of amino acids. One of the rRNA acts as a catalyst for peptide bond formation, which is an example of RNA enzyme (ribozyme). Translation is a process that has evolved around RNA, indicating that life began around RNA. Since, transcription and translation are energetically very expensive processes, these have to be tightly regulated. Regulation of transcription is the primary step for regulation of gene expression. In bacteria, more than one gene is arranged together and regulated in units called as operons. Lac operon is the prototype operon in bacteria, which codes for genes responsible for metabolism of lactose. The operon is regulated by the amount of lactose in the medium where the bacteria are grown. Therefore, this regulation can also be viewed as regulation of enzyme synthesis by its substrate.
Human genome project was a mega project that aimed to sequence every base in human genome. This project has yielded much new information. Many new areas and avenues have opened up as a consequence of the project. DNA Fingerprinting is a technique to find out variations in individuals of a population at DNA level. It works on the principle of polymorphism in DNA sequences. It has immense applications in the field of forensic science, genetic biodiversity and evolutionary biology.
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