• The genome of an organism is the complete DNA content, which includes DNA present in the nucleoid in prokaryotes and the DNA present in the chromosomes of the nucleus, mitochondria and plastids in eukaryotes.
• There are two different approaches of genome mapping, physical mapping and genetic mapping.
• A genetic map estimates genetic distances between genetic loci, which are responsible for a set of well-known phenotypes. Genetic distances are measured in centimorgan or map unit (a unit to measure genetic linkage of two phenotypes).
• Physical mapping is based on map features, such as restriction enzyme sites (restriction mapping) and sequence tagged sites (STS).
• In restriction mapping, restriction enzymes cut DNA at a specific base sequence, resulting in fragments of DNA that can be visualised by agarose gel electrophoresis in which DNA fragments are separated based on their sizes.
• STS are unique DNA sequences (200–500–bp) with well–known location, these serve as a useful landmark in the creation of a physical map of a genome. STS can be demonstrated by PCR.
First generation DNA sequencing technology was a multistep procedure involving separation of chromosomes, restriction digestion and ligation to high capacity cloning vectors.
• First generation DNA sequencing technique used a method of sequencing by chain termination, where a ladder of single stranded DNA with specific fluorescence tagged terminating bases are separated and detected by a fluorescence detector. By this method, base position and type of DNA bases are identified.
• Next generation DNA sequencing technology depends on advance computing algorithms to take millions of short sequence outputs, assembling in large contigs, representing DNA sequences of large genomes.
• Next generation sequencing includes techniques, like whole genome sequencing (WGS), Targeted resequencing, clinical exome sequencing, etc., and Chipseq, RNAseq are some of the advance applications of sequencing technologies.
• Nanopore sequencing technology uses two proteins: DNA helicase and porins like molecules to bind, unwind double stranded DNA and push single stranded DNA through porins like molecules to identify base pattern. This technique is simple, rapid, cost efficient, and displays results in real-time, and useful for genotyping and high mobility testing.
• Metagenomics involves sequencing of DNA or cDNA present in a microbial community.
• Computational genomics involves assuage of high performance computing clusters and workstations to analyse genomics data.
• Genome engineering is a technology to modify a genome (such as inactivate, delete, integrate, transduce and edit the genome).
• Transposons are DNA elements with the ability to move from one position of DNA to another, and can be exploited to engineer a knockout of the existing genes.
• Genome editing using CRISPR-Cas9 (use guide RNA along with Cas9 endonuclease) involves double strand break, homologous DNA repair to generate the edited DNA sequence.
• Structural genomics includes the study of structural organisation of DNA region in chromosome and nucleosome status of genome.
• Comparative genomics identify a set of common genes that form core genome and other genes that are unique to a species. So, it is a starting point for genome based taxonomy and phylogenetic lineage study.
• Functional genomics aims to study the physiological and pathological function associated with the state of a cell
• Protein engineering is applied to develop novel reagents, diagnostics and therapeutics. It is used to develop recombinant protein with 6 histidine-tag (6-His-tag) for the ease of purification and to develop fluorescent proteins to track the cellular localisation of proteins.
• Protein engineering technology is also involved in the production of humanised monoclonal antibody, single chain antibody and a recombinant immunotoxin which is an engineered antibody that delivers toxin to specific cellular targets.
