Authors: Dr. Rajesh C. Jeeterwal*1 and Anju Nehra2
1Young Professsional- II, ICAR-AICRP on Pearl Millet, Mandor, Jodhpur 342304 (Rajasthan)
2Senior Research Fellow, Agriculture Research Station (Agriculture University, Jodhpur), Mandor, Jodhpur 342304 (Rajasthan)
*Corresponding author email: firstname.lastname@example.org
Gene cloning refers to the assembly of an oversized population of a DNA fragment in pure form. Gene cloning involves the formation of a DNA and its introduction into an acceptable host, resembling E. coli or Bacillus subtilis. The host used ought to be without plasmids. The walls of host bacterium are created permeable by treatment with calcium chloride or a lysozyme. The DNA is additional to the culture within which such host bacterium are growing. The DNA is concerned by the bacterium together with the nutrients. It replicates whenever the bacterial cell divides. The bacteria E. coli divides each twenty two minutes underfavourable conditions, theoretically giving rise to a billion cells in under eleven hours. This may produce an outsized clone of recombinant deoxyribonucleic acid.In some cases, the bacterial cells additionallytranscribe the DNA molecules into messenger RNA, which is, in turn, translated into peptide chains. Hence, cloning may be utilized not solely to produce large quantities of the deoxyribonucleic acid sequence of an individual gene however additionally large amounts of the genes protein product furthermore.
Opening up a new horizon of research in genetics, gene cloning or the recombinant DNA (rDNA) technology, made a sensational beginning during 1970s. The first rDNA product was Somatostat in, a growth hormone produced by Itakura et al. in 1977. With sky being the limit, this technology has the most diversifying applications which include production of food and other supplements, diagnosis of infection and different genetic diseases, prophylaxis and therapy against diseases, biofertilizers, pesticides, metal extractions, pollution control etc. It is within the realm of gene from any living entity can be transferred into another life forms, be it a bacteria, yeast or fungi or mammal and the gene can be amplified immensely and the gene product harvested in leaps and bounds.
Methods of cloning and menipulation
The mechanics of gene manipulation dictates identification and isolation of the gene of interest, putting it in a host vector like bacteria or yeasts etc. and amplifying the gene along with the vector inside the host. The isolated DNA from donor source is restricted by Type-II restriction endonucleases. These enzymes recognize mostly a six base pair pallindromic DNA sequence (which reads same from either ends of complementary strand) and cuts within the sequence. The restriction fragments are ligated in a vector DNA which has been restricted earlier with the same restriction endonucleases. These restrictions leave the DNA with identical protruding ends, which are also known as sticky ends or staggered ends or cohesive ends.
The restriction fragments thus generated in the donor DNA are mixed with vector DNA and ligated by the enzymes DNA ligase under appropriate conditions. The ligated passenger and vector DNA are then passaged into competent host cells by the process called bacterial transformation. Competence of E.coli is achieved by treatment with calcium chloride. It can also be performed by electrical treatment called electroporation, which increases the transformation efficiency by about 1-20 times. Laser beam is also an effective method of developing competence. It is possible to transform E.coli to as high as 10 10 transormants per microgram of plasmid DNA. This method of ligation of all the restricted donor DNA to vector DNA and transformation is called SHOTGUN CLONING. This method besides requiring screening of large number of recombinant clones runs the risks that the gene may be ligated in a reverse orientation with respect to the promoter thus gene expressions is not possible. This problem can be avoided by cutting the foreign DNA as also the vector DNA with two restriction enzymes. This ensures proper orientation of the insert DNA upon ligation. This also prevents re-circularisation of vector as well as passenger DNA for want of complementarities. This method of cloning is called DIRECTIONAL CLONING. Looking at the nut bolts levels of genetic engineering it will be seen that the cohesive ends bind specifically with complimentary base pairs produced on the vector DNA cut by the same enzyme. However, there are few enzymes, which make blunt end cut which can be ligated by DNA ligase obtained from bacteriophage T4 under optimum ATP concentrations. Gene cloning can be performed in E.coli, Bacillus subtilis (for extrasecretory functions) yeast/eukaryotic (extrasecretory), plant cells and mammalian cells. Ligation of passenger DNA can be done on plasmid (upto 10 kilobase inserts) or in lamda bacteriophase (upto 25 kb) and in cosmid (upto 45 kb) vector. Wide diversity is available today in all the above kinds of vectors, each having some advantage over the other. Thus a proper vector has to be selected before initiating any cloning experiment.
The transformed cells are later cultured in broth or on agar plates so that the inserted gene multiplies several folds. Gene cloning when performed in plasmid vector, a high copy number plasmid is selected for getting more copies (20-700) of the inserted gene. These plasmids are called RELAXED PLASMIDS and their replication is independent of replication of host chromosome (e.g. Col E1, pMB, pUC etc.). On the contrary the plasmid whose replication is dependent with the replication of host chromosome yield few copies (1 or few) per cell. These are called the plasmids with stringent replications or stringent plasmids (e.g. pSC 101). The number of relaxed plasmids can be increased further upto 5000 copies per cell by a method called PLASMID AMPLIFICATIONS. Plasmid amplification involves growing the culture further with vigorous shaking in presence of protein synthesis inhibitor like chloamphenicol, tetracycline or spectinomycin. The amplified recombinant vector is then isolated and the inserted gene is isolated by restriction with the same restriction endonucleases used earlier. The gene isolated in abundance can be used for sequencing, hybridization, cell free translation or any other purpose. This is the method of in-vivo gene amplification and was the only method of gene amplification till the development of polymerase chain reaction (PCR), which is method of in-vitro DNA amplification, developed in 1980s.
Let us now consider the steps, which involve great intricacies. To begin with it is most important to identify the gene of interest, which has to be cloned.
IDENTIFICATION OF THE GENE INTEREST
An important step in cloning and amplification of required gene involves identification and isolation of the gene to be cloned. Once the gene has been identified it can be isolated by any of the following methods:
- Cleavage by restriction endonucleases
- Mechanical shearing
- RNA directed synthesis
- Chemical synthesis For identification of the gene either nucleic acid probe or the protein (for use as an antigen) or an antibody to the product of gene is required. The nucleic acid (DNA or RNA) probe identifies the gene, which can be cloned. The product of the gene, say a protein can be used to generate antibody of the protein (antigen). The antibody can be utilized for identifying the translating m-RNA producing growing polypeptide chains and nascent protein (antigen) molecules on a series of ribosome (polyribosomes or polysome). The method is known as POLYSOME PRECIPITATION. If the relevant nucleic acid, antigen or antibody is not available, the same from related species or even a distantly related species may be tried. The eukaryotic m-RNA which has a poly-A tail (except for histone, interferon and immunoglobin) can be isolated by affinity chromatography through a poly dT cellulose matrix. Purification of m-RNA is facilitated considerably as it has a characteristic base composition. In silk fibrin and collagen for instance, the known periodic amino acid sequences predict a special composition of the corresponding m-RNAs. These m-RNAs are easily separated in calcium chloride density gradient centrifugations by virtue to their high guanine content and are relatively large (32 and 27s, respectively) in size. The other methods of identifying a gene are a direct selection. In this case identifications of the gene is not made before cloning, but selection is made after cloning of all the restriction fragments (shotgun cloning) on different selective media plates. There are some genes, which are also directly selected as in some cases only the recombinants grow on the selective plates. This provides direct selection of the gene of interest.
After the gene has been identified and isolated, the next step is to ligate it with a vector and put in the appropriate host for multiplications to high copy number. The cloning vector could be plasmid (extra chromosomal DNA), a bacteriophage (lamda, baculo virus, adeno virus, retro virus), cosmid (a combination of plasmid and phage), yeast, phage M12, T1 plasmid, phasgemids, plants and fungi.
APPLICATION OF PLANT GENE CLONING TECHNIQUES
- Single gene transfer
- Mass gene transfer
- Soma clones
- Plant cell – Bacterium, Somatic hybrid
- Pathogen resistance
- Resistance to herbicides
- Homozygous cultivars
- Enrichment of storage protein
- An introduction to photosynthetic capacity
- Stress resistance
- Post harvest preservation
- Selection of autotrophs
- Secondary metabolic analysis
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