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Steps and strategies of gene cloning

A gene is the fundamental unit of heredity, composed of DNA and providing instructions for protein synthesis. Gene cloning involves creating identical copies of a gene of interest by integrating it into a vector (like plasmids) and introducing it into host cells. Key steps include generating foreign DNA, vector selection, insertion into the vector, and screening recombinant clones.

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A gene is a basic physical and functional unit of heredity. Genes which are made up of DNA, acts as instructions to molecules called proteins.
Cloning is the process of generating an identical copy of a cell or an organism. Clones are those organisms that have identical genes.
“Gene cloning is genetic engineering technique in which a gene of interest is fused into a plasmid and introduced into a suitable host, self replicates and generates a large number of identical copies of the particular gene.”
STEPS INVOLVED IN GENE CLONING: Generation of foreign DNA molecules Selection of suitable vector Insertion of foreign DNA into vector Recombinant DNA introduced into host cells Selection and screening methods of recombinant clones
Strategies to generate foreign DNA molecules: Using restriction enzymes cDNA synthesis Using gene machine
USING RESTRICTION ENZYMES:  Restriction enzyme is a dna cutting enzyme that recognizes specific sites in DNA, they are often called as restriction endonuclease.  It cleaves the dna in two forms. They are i. Blunt end ii. cohesive end
Eg: Eco K1 Eco A1 Cfr A1 Eg: Eco R1 Bam H1 Hind III Eg: Eco P1 Hinf III EcoP15i
cDNA SYNTHESIS:  Reverse transcription is the process of making a double stranded DNA molecule from a single stranded RNA template catalyzed by an enzyme reverse transcriptase.  it is also called as RNA-dependent DNA polymerase.
USING GENE MACHINE: “Gene machine is a fully automated commercial instrument, also called as automated polynucleotide synthesizer which synthesizes predetermined polynucleotide sequence.”
Working principle: “Development of insoluble silica-based support in the form of beads which provides support for solid phase synthesis of DNA chain.” “Development of stable deoxyribonucleoside phosphoramides as synthons which are stable to oxidation and hydrolysis, and ideal for DNA synthesis.”
vector Vector is a extra chromosomal double stranded circular DNA which has the ability to self replicate. It acts as a carrier molecule in which foreign DNA can be inserted or integrated and replicates in the host cell to produce multiple clones of recombinant DNA.
VECTOR SIZE: The plasmid size less than 10 kb is desirable for a cloning vector. Plasmids ranges from 1.0kb to 250kb,but only few are used for cloning. Selection of suitable cloning vector
Copy number:  The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell.  Based on copy number plasmids are classified into two types: i. Stringent plasmids – high copy number ii. Relaxed plasmids – low copy number
Selectable marker:  The selectable marker gene permits the selection of host cells which bear recombinant DNA (transformants) from those which do not bear rDNA(non-transformants).  Two types of selectable markers 1. Drug-resistance markers 2. Auxotrophic markers
Restriction sites:  Restriction sites allow cleavage of specific sequence by specific restriction endonuclease to insert the desired DNA fragment.  Restriction sites in E.coli cloning vector includes Hind III, BamH1, EcoR1, Sal1, PvuI, CIaI etc.
ORI: Origin of replication(ORI) is a specific sequence of nucleotide in DNA is linked to this sequence then along with vector replication, foreign DNA also starts replicating within host cell.
Steps and strategies of gene cloning