1. RECOMBINANT DNA TECHNOLOGY

2. Scientists have developed a number of biochemical and genetic techniques by which DNA can be separated, rearranged, and transferred from one cell to another. The discovery of restriction enzymes and many other enzymes which can be useful as tools in gene manipulation have brought in vitro recombining of DNA to a reality.

3. I. Enzymes commonly used in recombinant DNA technology 1. Restriction endonucleases (RE) Restriction enzymes are found in bacteria. Bacteria are always exposed to a danger of being killed by phage (viruses that infect bacteria). In 1972, for the first time RE was used to cut the DNA specifically and fragments were ligated to first recombinant molecule. Restriction endonucleases are hydrolases and cleave phosphodiester bonds of double stranded DNA at specific palindromic sites within the chain to produce 5 1 PO 4 and 3 1 OH ends. They are called as “restriction endonucleases” mainly due to their natural function in restricting the growth of the virus that attack bacteria

4. There are three types of restriction endonucleases : Type I, II and III. Type I and III recognize specific nonpalindromic sequences in the DNA chain, cleave the chain at different sites away from the recognition site, thus producing DNA fragments of different length and ends. Type II restriction endonucleases, recognize specific palindromic sequences that range generally from 4–8 nucleotides and cut the chain within the site, thus producing specific DNA fragments with known ends ↑

5. 2. DNA polymerase DNA polymerases synthesize complementary nucleotide sequence on a template nucleotide strand. DNA polymerase I, isolated from E. coli, synthesizes a complementary strand on a template DNA in 5 1 → 3 1 direction. It also possess low level of exonuclease activity in both 5 1 → 3 1 and 3 1 → 5 1 directions. This enzyme is used in labeling of DNA to prepare probe.

6. Klewnow enzyme is the large fragment of the DNA Polymerase I of E. coli. It possess 5 1 → 3 1 polymerase activity and 3 1 →5 1 exonuclease activity but lacks the exonuclease activity in 5 1 → 3 1 direction. Tag DNA polymerase is isolated from a bacterium Thermus aquaticus, living in hot springs and active even at 94ºC. This enzyme is highly thermostable for which it is used for DNA amplification during polymerase chain Reaction (PCR). It does not have 3 1 → 5 1 exonuclease activity and hence cannot carry out proof reading

7. 3. Reverse Transcription of mRNA Recently, a superior method of selecting desired genes has been discovered, which is called reverse transcription of mRNA. The m-RNA is mixed with the enzyme “reverse transcriptase”. This enzyme was found in some viruses having RNA as genetic information instead of DNA. The virus utilizes reverse transcriptase to catalyze the reverse process of synthesizing a complementary DNA chain on an RNA template, and to insert it into the chromosome of the host cell

8. 4. DNA ligase Two DNA fragments from the same or different sources can be joined to produce a recombinant DNA. DNA ligase performs this function by catalyzing the formation of phosphodiester bond between adjacent 3 1 hydroxyl and 5 1 phosphate termini of the two different DNA fragments. DNA ligase produced by T 4 bacteriophage is used, which needs ATP and Mg++ for its activity. T 4 DNA ligase can ligate (join) both double stranded DNA and single stranded DNA fragments, DNA fragments having compatible staggered ends, and those with blunt ends. The rate of ligation of compatible staggered ends is 50 – fold higher than that of blunt ends. It repairs nicks in double stranded DNA molecules.

9. II. VECTORS Vectors are the carrier DNAs into which ‘foreign’ DNAs or genes of interest are spliced to make a recombinant DNA. Vectors along with this ‘foreign’ DNAs (i.e. recombinant DNA) are then introduced into appropriate host cell and are maintained for study or expression. Organisms with chimeric property can be produced with the help of cloning vectors. There are two types of vectors. cloning vectors and expression vectors.

10. a.Cloning vectors Used for obtaining millions of copies of cloned DNA segment. The cloned genes in these vectors are not expected to express themselves at transcription or translational level. Cloning vectors are used for creating genomic library or preparing the probes or genetic engineering experiments or other basic studies. Most cloning vectors were originally derived from naturally occurring extrachromosomal elements such as bacteriophages and plasmids.

11. b. Expression vectors Allow the expression of cloned gene, to give the product (protein). This can be achieved through the use of promoters and expression cassettes and regulatory genes (sequences). Expression vectors are used for transformation to generate transgenic plant, animal or microbe where cloned gene expresses to give the product. Commercial production of product of cloned gene may also be achieved by high level expression using the expression vectors.

12. 1.Plasmids The occurrence of plasmids in E. coli came to light in the early 1950’s through the pioneering work of Joshua Lederberg in the USA and William Hayes in England. Plasmids are genetic elements that are stably inherited without being a part of the chromosome (s) of their host cells. They are found in bacteria and fungi of many kinds but not in higher eukaryotes and are not essential to the survival of the host cell. They may be composed of DNA or RNA and may be linear or circular. Double-stranded DNA plasmids appear to exist as predominantly covalently closed circular molecules in bacterial cells. Both circular and linear plasmids are found in yeast and other fungi.

13. Linear yeast plasmids composed of either RNA or DNA, can encode protein toxins that inhibit the growth of sensitive yeasts. The number of molecules of a plasmid found in a single bacterial cell is termed as copy number The smallest bacterial plasmids are about 1.5 kb and the largest are greater than 1500 kb. The vast majority are circular. However, several very large linear DNA plasmids, up to 500 kb long, have been found in species of Streptomyces and Nocardia. Smaller plasmids are much desirable for gene cloning experiments. Larger plasmids are less in number whereas smaller ones are more in number. Plasmids with larger copy number are more useful for gene cloning experiments. Plasmid PBR 322 is derived from transposon Tn 3, plasmid pMBI, and plasmid pSC 101. pMBI- replicon,Tn 3 - ampicillin transposon, pSC 101 – tetracycline resistance region.

14. 2. Bacteriophages as cloning vectors Phage has a linear DNA molecule. So a single break creates two fragments. Foreign DNA can be inserted between them and two fragments can be joined. Such phages when undergo lytic cycle in host will produce more chimeric DNA. Wild type lambda phage could accommodate only 2.5 kb of foreign DNA. Phage vectors are restructured by removing nonessential genes and making vector DNA smaller so that larger insert can be accommodated in phage head during packing. Lambda phage such prepared has one Eco RI site and accommodates 20-25kb of foreign DNA. They are used for preparing genomic library of eukaryotes

15. 3. Cosmids Cosmids are the novel cloning vectors which possess properties of both plasmid and phage. Cosmids were first developed in 1978 by Barbara Hohn and John Collins. Cosmids contain a cos site of phage (which is essential for packaging of nucleic acid into protein coat) plus essential features of plasmid (such as plasmid origin of replication, a gene for drug resistance) and several unique restriction sites for insertion of DNA to be cloned. Cosmids can be perpetuated in bacteria in plasmid form, but can be purified by packaging in-vitro into phages. Advantage of using cosmid vector is that larger DNA can be cloned than what is possible with phage of plasmid.

16. 4. Yeast Cloning Vectors Yeast cloning vectors are the carrier DNA molecules into yeasts. Yeasts are eukaryotes. Yeast artificial chromosome (YACS) are most sophisticated yeast vectors. They have centromeric and telomeric region of a chromosome. These regions are needed to allow chromosome to be replicated in yeast cells. Due to origin of replication that is present, replication of DNA occurs. These elements are placed in single DNA fragment which can be used as vector to clone foreign DNA into yeasts. The advantages of YAC include very large piece of DNA can be cloned. Only single copy of YAC is present per cell.

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