The fundamental unit of information in living systems is the gene. Genome is the set of all genes and genetic signals of a cell. Gene is expressed through a sequence of events. The central dogma of molecular biology, comprises the three major processes replication, transcription and translation. The genetic message encoded in messenger RNA (mRNA) is translated on the ribosomes into a polypeptide with a particular sequence of amino acids. An RNA base sequence (a set of 3 bases) corresponding to a particular amino acid is called a codon. The genetic code is the set of all codons. The genetic code is a triplet code, and all 64 possible codons carry information of some sort. The code is highly redundant. Polypeptides are synthesized from the amino terminus toward the carboxyl terminus, by adding amino acids one by one to the carboxyl end. An important feature of intiation of polypeptide synthesis in both prokaryotes and eukaryotes is the use of a specific initiating tRNA molecule.
Mutation is the process by which the sequence of base pairs in a DNA molecule is altered. Mutations can be divided into base pair substitution mutation and base pair insertion or deletions.
Frame shift mutation usually results in a nonfunctional protein. An addition or deletion of one base pair, for example, shifts the mRNA’s downstream reading frame by one base, so that incorrect amino acids are added to the polypeptide chain after the mutation site. Point mutations are single base changes, that do not affect the reading frame, that is, the mutation only makes a single change in a single codon,
Mutations can also be defined according to their effects on amino acid sequences in proteins. They are missense mutation, silent mutation, nonsense mutation. Forward mutations change the genotype from wild type to mutant and reverse mutations (or reversions or back mutations) change the genotype from mutant to wild type or to partially wild type.
The term mutant refers to an organism in which either the base sequence of DNA or the phenotype has been changed. The process of formation of mutant organism is called mutagenesis.
DNA fingerprinting is the present day genetic detective in the practice of modern medical forensics. The underlying principles of DNA fingerprinting are briefly described.
The structure of each person’s genome is unique. The only exception being monozygotic identical twins (twins developed from a single fertilized ovum). The unique nature of genome structure provides a good opportunity for the specific identification of an individual. The DNA fingerprint is an analysis of the nitrogenous base sequence in the DNA of an individual.
The original DNA fingerprinting technique was developed by Alec Jaffreys in 1985. Although the DNA fingerprinting is commonly used, a more general term DNA profiling is preferred. This is due to the fact that a wide range of tests can be carried out by DNA sequencing with improved technology.
The amount of DNA required for DNA fingerprint is remarkably small. The minute quantities of DNA from blood strains, body fluids, hair fiber or skin fragments are enough. Polymerase chain reaction is used to amplify this DNA for use in fingerprinting. DNA profiling has wide range of applications – most of them related to medical forensics. Some important ones are listed below.
• Identification of criminals, rapists, thieves etc.
• Settlement of paternity disputes.
• Use in immigration test cases and disputes.
In general, the fingerprinting technique is carried out by collecting the DNA from a suspect (or a person in a paternity of immigration dispute) and matching it with that of a reference sample (from the victim of a crime, or a close relative in a civil case).
Ames test is an indicator of whether the chemical is a mutagen. The Ames test assays the ability of chemicals to revert mutant strains of the bacterium Salmonella typhimurium to wild type. The most commonly employed gene transfer methods are transformation, conjugation, transduction, electroporation, lipofection and direct transfer of DNA. Transformation is genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings. Competence refers to the state of being able to take up exogenous DNA from the environment. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges (e.g. pilus) and transfer single stranded DNA (from donor to recipient). Transduction is the transfer of bacterial genes from one bacteria to other by viruses, e.g. Bacteriophage (Bacterial viruses). Recombination is the process in which one or more nucleic acids molecules are rearranged or combined to produce a new nucleotide sequence. Cloning in the molecular biology sense (as opposed to cloning whole organisms) is the making of many copies of a segment of DNA, such as a gene. Cloning makes it possible to generate large amounts of pure DNA, such as genes, which can then be manipulated in various ways
Vectors are the DNA molecules, which carry a foreign DNA fragment to be cloned. They are cloning vehicles, examples of which are Plasmids, Bacteriophages, cosmids, phagemids and artificial chromosomes. Bacterial plasmids are extra chromosomal elements that replicate autonomously in cells. They are viruses that replicate within the bacteria. Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage. Phagemids are the combination of plasmid and phage, and can function as either plasmid or phage. The plasmid vectors that are specifically designed to replicate in two or more different host organisms (say in E. coli and yeast) are referred to as shuttle vectors. An expression vector is a cloning vector containing the regulatory sequences (promoter sequence) necessary to allow the transcription and translation of a cloned gene or genes. Restriction enzymes are the bacterial enzymes that recognize a specific base sequence in a DNA molecule (from any source) and make two cuts one in each strand generating 3′ – OH and 5′ – P termini.
There are several techniques used in recombinant DNA technology or gene manipulation. The most frequently used methods are agarose gel electrophoresis, isolation and purification of nucleic acids, nucleic acid blotting techniques, DNA sequencing, chemical synthesis of DNA, gene transfer methods, polymerase chain reaction, construction of gene library, radiolabeling of nucleic acids etc. Gel electrophoresis is a routinely used analytical technique for the separation and purification of specific DNA fragments. PCR is a cell free amplification technique. The three – step cycle is repeated to obtain copies of target DNA in large numbers.
The DNA markers are highly useful for genetic mapping of genomes. RFLPS (Restriction Fragment Length Polymorphisms), VNTRs (mini satellites or Variable Number Tandem Repeats), STRs (Microsatellites or Simple Random Repeats), SNPs (Single Nucleotide Polymorphisms) are types of DNA sequences (stretch of DNA) which can be used as markers. These markers are used in disease diagnosis and DNA fingerprinting.