Molecular Biology. A Practical Guide - Velikov V.A. 2013

Gene Cloning

Gene cloning involves obtaining a bacterial or Yeast clone containing recombinant DNA, from whose Cells the DNA of a specific gene and its product (the target protein) can be isolated in significant quantities. Gene cloning forms the foundation of Introduction/32.html">Genetic Engineering, or Recombinant DNA technology, as it was coined by its pioneers. While the Structure/179.html">Practical Applications OF this technology are well known, its significance for fundamental science is equally profound, highlighting the collinearity between DNA and protein. This technique is originally referred to as molecular cloning because a specific DNA molecule isolated from The Genome directs the synthesis of only a single type of protein molecule. Compared to genomic DNA, it represents an individual molecular clone—a distinct "molecular machine" for protein production. In Gene Therapy or the generation of Transgenic Plants and animals, researchers invariably employ genes initially cloned in bacterial or yeast cells. Recombinant DNA was first successfully produced by Paul Berg and colleagues (Berg et al., 1972).

Gene cloning is carried out by inserting the gene into a vector. A vector is a circular DNA molecule (derived from a plasmid, virus, or bacteriophage) capable of autonomous Replication within a chosen host Cell system. The gene DNA and vector DNA, digested with the same restriction enzyme to generate complementary "sticky ends," are covalently joined into a single circular molecule using the enzyme DNA ligase. The resulting recombinant DNA is introduced into bacterial cells, where it can replicate autonomously—independently of the chromosome and in high copy numbers. For instance, the pBluescript II phagemid can be present at about 300 copies per cell in an E. coli population. By cultivating the Cells of the resulting producer clone, the desired protein can be obtained in unlimited quantities. By definition, this constitutes an inexhaustible resource. The term "superproducer" highlights the exceptionally high level of target Protein Synthesis achieved by the host cell.

The cloning strategy for each specific gene must be tailored individually. A properly devised strategy accounts for half the success (encompassing vector and restriction enzyme Selection, selection systems, clone screening, preliminary gene isolation, and much more). The cloning Procedure can be broken down into several stages:

* Preparation of Vector DNA (restriction digest),

* preparation of gene DNA (restriction digest, PCR, or gene synthesis),

* ligation ("joining") of the gene DNA and vector DNA,

* transformation of E. coli cells with the ligation reaction products,

* selection of transformed E. coli clones,

* screening of selected clones to confirm the presence of the gene DNA insert within the vector,

* validation of expression for the cloned gene.

A general schematic of molecular cloning is presented in Appendix 14.

The advent of PCR significantly streamlined the gene cloning process, as it allows for the isolation and Amplification of the target gene's DNA prior to the ligation stage with vector DNA, eliminating the need to screen for the desired clone within an Organism's gene library hosted in E. coli—a formidable task that demands an experienced researcher. In essence, PCR generates millions of molecular clones of a specific DNA segment, except that this DNA cannot replicate further, and most importantly, cannot be expressed without being incorporated into a vector and introduced into a cell.

Genes with a known (!) Primary Structure can also be "assembled" in vitro from synthetic oligonucleotides. For PCR, it is only necessary to know or accurately predict The nucleotide sequences of the primers.