Blunt end ligation:
In this technique a restriction enzymes is used to cut a duplex DNA at the same site in both the strands.
The broken ends are then used for joining with the two ends of foreign DNA molecule irrespective of the sequences present at the broken ends of the two molecules. The blunt end ligation is made possible by the use of the T4 DNA lygase. This technique also has a disadvantage in that the broken ends of the same DNA molecule may join together.
Construction of genomic and cDNA libraries:
In order to isolate required gene or genes from a genome a mixture of clones each carrying DNA fragments derived either from the natural genome or from cDNA is prepared. This mixture contains thousands of clones and is collectively called genomic library.
Similarly when cDNA clones are collected it is known as cDNA library.
Construction of genomic library:
Cloning an entire genome in the form of fragments mixed randomly is known as genomic library. This may be done by a method known as shotgun method (see earlier in the same chapter for details). With a genome library clones can be perpetuated indefinitely in a plasmid vector and retrieved whenever necessary. Genomic libraries can also be prepared by using a number of restriction endonucleases one at a time so that one will have fragments of different sizes with cuts at different places.
This has a disadvantage in that many a time the incision site many are within a gene so that complete genes may be difficult to obtain. This may be overcome by using restriction enzymes which have very short recognition sequences so that the fragments that are obtained will be long and the chances of splicing within a gene are avoided.
It has already been explained above as to how to obtain a cDNA using its complimentary mRNA under the influence of the enzyme reverse transcriptase. When once a cDNA molecule is obtained it can be made double stranded by the use of DNA polymerase a collection of such cDN a molecules will constitute cDN a library.
Screening of the library when a genomic or cDN a library is available it can be used for the isolation of a specific gene sequence. For this the DNA library has to be screened. This is done with the help of a technique known as Colony hybridization. In these technique bacteria carrying chimeric plasmids are cultured into colonies and are then lysed on nitrocellulose filters. Their DNA is denatured in situ and fixed on the filter which is then hybridized with a radio actively labeled probe carrying a sequence related to the gene to be isolated.
In other words, the probe will have nucleotides complimentary to the ones present in the gene to be isolated. Colonies that carry the sequence will be identified by dark spots after radio autography so that the colonies carrying the vector with the desired gene sequence can be recovered from the master plate and used for further experiment.
Transfer of the recombinant DNA into a cloning organism:
When once a mixture of recombinant DNA is obtained it has to be transferred to a suitable cloning organism for the purpose of expression. Several techniques are used to transfer the recombinant DNA into suitable cloning cells.
Transforming the cloning cells with recombinant DNA is a difficult process as the cells of bacteria (E.coli) have restriction enzymes which will immediately degrade the foreign DNA. To escape from degradation exponentially growing culture of E.coli is pretreated with CaCl, at low temperature and thereafter the DN A is mixed up. This treatment is known to sufficiently prevent the action of restriction enzymes.
When the recombinant plasmids get established in the cells of E.coli the cells are said to be transformed. Instead of plasmids even X phages could be used as transforming elements. The entry of the phage into E.coli is usually termed transfection (a combination of transformation and information). Not all the cells will accept recombinant DNA and get transformed. It has been estimated that about 105 transformers per microgram of cloned circular plasmid can be generated.
The cells which have been transformed will grow into colonies along with other non transformed ones. Colonies with transformed cells can be identified with suitable techniques.
Expression of cloned DNA:
The purpose of genetic engineering is not just to separate and clone the genes in a suitable organism. Isolation and multiplication of genes is only a means ultimately to obtain the gene product namely the required protein. Hence the recombinant DNA molecules should be allowed to express in the new environment (E.coli cells) and produce whatever protein they were producing. For instance if an insulin gene from human beings is transferred into E.coli the gene should be able to produce insulin molecules using the protein synthesizing machinery of the bacterium Expression of a cloned gene is a very difficult process.
This is more so when a eukaryotic gene is inserted into a prokaryotic organism. As the protein synthesizing genetic machinery of prokaryote is different from eukaryotes, the eukaryotic gene should be provided with the necessary ingredients of a prokaryote to produce the protein the factors that are necessary for eukaryotic gene to express in the prokaryotic environment are as follows: (i) Supply of prokaryotic promoter necessary for expression of eukaryotic gene (ii) Supply of ribosomal binding sites for the cloning vector (iii) Removal of introns from mRNA obtained from the eukaryotic genes (iv) Inhibition of bacterial gene responsible for degradation of the protein produced by the eukaryotic gene.
Recovery of the gene product:
This is the final step in genetic engineering techniques. After all the hurdles are passed and the eukaryotic gene has successfully expressed itself in the prokaryotic cell, the required product namely the eukaryotic protein is produced. This can be identified by antigen antibody reaction (for details see genetically engineered insulin later in the same chapter). The protein produced can be purified by standard chemical processes.