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In this article we will discuss about the gene isolation and cloning of DNA.
The formation of new combinations of genetic material by the insertion of nucleic acid produced outside the cell into a virus, bacterial plasmid or any other vector system to allow its incorporation into a host organism in which it is capable of continued replication and expression is termed as genetic engineering.
The techniques involved are also referred to as gene cloning, in vitro genetic manipulation or recombinant technology. The transfer of genetic material between individuals takes place in nature by conjugation, transduction and transformation.
Whatever the long-term aim of a molecular biology project the initial objective is usually to isolate a single gene or other specified DNA segment from the organism being studied. This is the essential preliminary step to DNA sequencing, to examine the expression profile of a gene, or for purification of the protein coded by a gene.
This initial objective can be achieved in either of two ways:
i. By DNA cloning, which involves insertion of the desired DNA molecule into a cloning vector followed by replication of the cloning vector in a culture of E. coli bacteria?
ii. By PCR, which involves enzymatic amplification of the desired DNA fragment?
‘Gene Cloning’ is a method of isolating a specific region of DNA and producing millions of identical copies of the DNA (gene) within a microbial cell culture.
The basic steps in a gene cloning experiment are:
i. Isolation and purification of DNA
ii. Cutting of DNA in required sites by the same restriction enzymes.
iii. Joining (ligation) the required piece of DNA to a vector
iv. Implanting the vector into the host cell by transformation
v. Selection of the transformants
vi. The recombinant DNA molecules replicate within the host cells.
vii. When the host cell divides, copies of the recombinant DNA molecules are passed on to the progeny.
viii. Continued replication of the host cells results in clones, each of which consists of identical cells all containing copies of a single recombinant DNA molecule.
This series of manipulations results in a clone library, comprising many different clones, each carrying a different segment of the original DNA. The next step is therefore to identify the clone that contains the gene of interest PCR in outline—PCR is a very different approach to isolate DNA segment.
Rather than a lengthy series of manipulations involving living cells, PCR is a test-tube reaction that is carried out simply by mixing together the appropriate reagents and incubating them in a thermal cycler, a piece of equipment that enables the incubation temperature to be varied over time in a pre-programmed manner.
The basic steps in a PCR experiment are as follows DNA is prepared from the organism being studied and denatured by heating to 94°C. A pair of oligonucleotides is added to the DNA; the sequences of these oligonucleotides enable them to anneal either side of the gene or other DNA segment that is to be isolated, and the mixture is cooled to 50-60°C so that these oligonucleotides attach to their target sites.
A thermostable DNA polymerase is added together with a supply of deoxy-ribonucleotides and the mixture is heated to the optimal temperature for DNA synthesis. The cycle of denaturation- annealing-extension is repeated 25-30 times, with the number of newly-synthesised DNA molecules doubling during each cycle. This exponential amplification results in synthesis of a large number of copies of the DNA sequence flanked by the pair of oligonucleotides.
Basic techniques needed for cloning and PCR are:
1. Handling bacteria
2. Preparation of DNA
3. Preparation of RNA
4. Separating DNA and RNA by gel electrophoresis
5. Purifying DNA molecules from electrophoresis gels
6. Construction of recombinant DNA molecules
7. Introduction of recombinant molecules into host cells and recombinant selection
8. Cloning vectors.
Handling bacteria:
One of the most important practical skills you as a microbiologist are the ability to grow pure cultures of bacteria including cultures infected with bacteriophages. This is because most gene cloning experiments use the bacterium E. coli as the host organism. Even if the long term objective is to clone a gene in an organism other than E. coli the initial manipulations will be carried out in E. coli because of the ease with which this bacterium can be handled.
Preparation of DNA:
For many, the first real molecular biology to be attempted is purification of DNA from the organism being studied.
Preparation of RNA:
The preparation of pure, intact RNA is relatively difficult because of the ubiquity of contaminating enzymes that degrade RNA molecules. RNases present in the cells from which the RNA is being extracted can be inactivated during the purification procedure by a suitable RNase inhibitor, but considerable problems can be presented by glassware and solutions contaminated with RNases derived from skin secretions. These enzymes are very resistant to physical treatments some even withstanding autoclaving and their control can be a problem. RNA can also be used instead of DNA as the starting material for PCR.
Separating DNA and RNA by Gel Electrophoresis:
Separating DNA and RNA molecules of different sizes is achieved by electrophoresis in an agarose or, more rarely, polyacrylamide gel. Agarose gels are routinely run to estimate the sizes of DNA and RNA molecules, which enables the success of a preparative technique or enzymatic manipulation to be assessed. Agarose gel electrophoresis is also the preliminary to analysis of DNA molecules by techniques such as Southern hybridisation.
Purifying DNA molecules from electrophoresis gels:
In some areas of molecular biology there are a number of alternative procedures for achieving the same objective.
1. The purification of DNA molecules from gels is one such case. Frequently, an agarose or polyacrylamide gel will provide a band that can be confidently identified as containing a DNA fragment of interest.
2. The next step might be to excise the band and purify the DNA molecules prior to construction of recombinant DNA molecules and further study.
Construction of recombinant DNA molecules:
The first real step in a gene cloning experiment is construction of recombinant molecules by insertion of DNA fragments into vector molecules.
Introduction of recombinant molecules into host cells and recombinant selection:
Once recombinant DNA molecules have been constructed they must be introduced into E. coli cells. This is quite straight forward and standard technique for different types of vector.
Cloning vectors:
By now it should be clear that the choice of cloning vector is an important one. It should determine the nature of the procedure used to introduce the recombinant molecules into the host cells and it should specify the way in which recombinants are selected.
It is important to choose a vector that is suitable for the size of DNA fragments that you wish to clone, as some vectors are designed for relatively small fragments (5 kb and less) whereas others require fragments above a certain size.