1. Formation of Recombinant dna

A. Tools Used in the Formation of Recom­binant DNA. Three types of “biological tools” are used in the synthesis of recombinant DNA. These include enzymes, vehicle or vector DNA, and pas­senger DNA. Cultures of cells are prepared for obtaining passenger DNA and cells to be used as host.

1) Enzymes. Many kinds of specific enzymes are employed in genetic engineering. These include lysing enzymes, cleaving enzymes, synthesizing en­zymes, joining enzymes, and alkaline phosphatases.

● Lysing Enzymes. These are used to open up the cells to get DNA for genetic experiments. Lysozyme is commonly used to dissolve the bac­terial cell wall.

● Cleaving Enzymes. These are used to break DNA molecule. They are further of 3 kinds: (a) exonucleases, which cut off nucleotides from 5' or 3' ends of DNA molecule ; (b) endonucleases, which cleave DNA duplex at any point except the ends ; and (c) restriction en­donucleases, which cleave DNA duplex at specific points in such a way that single- stranded free ends project from each fragment of DNA duplex. These single-stranded free ends are called "sticky ends" because they can join similar complementary ends of DNA fragment from some other source. Restriction endonuclease was found by Arber in 1962 in bacteria. It acts as a molecular scissors or a chemical scalpel. Restriction endonuclease —1 (ECOR - 1) is found in the colon bacterium Escherichia coli. It recognizes the base sequence GAATTC in DNA duplex and cleaves its strands between G and A as shown below. The arrows indicate the sites where the enzyme cleaves the sequence.

3' - C T T A A G - 5'

5' - G A A T T C - 3'

● Synthesizing Enzymes. These play a role in the synthesis of DNA strands on suitable templates. They are further of 2 types : (a) reverse transcriptases, which help in the synthesis of com­plementary DNA strands on RNA templates ; (b) DNA polymerases, which aid in the synthesis of complementary DNA strands on DNA templates.

● Joining Enzymes. These help in sealing gaps in DNA fragments which are otherwise joined by complementary base pairing. Tu ligases are ex­amples. These act as a molecular glue. They join DNA fragments by forming phosphodiester bonds.

● Alkaline Phosphatases. These cut off phosphate group from the 5' end of linearised circular DNA to check its recircularization.

b) Vehicle DNA. The DNA used as a carrier for trans­ferring a fragment of foreign DNA into a suitable host is called vehicle DNA. It is also called cloning vector or gene carrier. The desired gene is in­troduced into a vector wherein recombinant DNA (r-DNA) is formed. The vector carrying r-DNA divides, thereby forming several copies of r-DNA. Five types of DNA are used as vehicles:

- plasmid DNA,

- bac­teriophage DNA,

- DNA of plant and animal viruses,

- transposons (jumping genes), and

- artificial DNA.

The first two are more commonly used vectors.

● Plasmids. The plasmids are small, circular DNA molecules present in bacteria in addition to the chromosomal DNA. They may pick up genes from one bacterium and transfer them to another. A plasmid having DNA of another organism integrated with it is known as cosmid, also recombinant plasmid, or hybrid plasmid.

● Bacteriophage DNA. The bacteriophages, or simply phages, are viruses that infect and kill bacteria. Their circular DNA is also suitable for use as a vehicle DNA.

c) Passenger DNA. It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. Three types of DNA are used as passengers : complementary, synthetic and random.

● Complementary or Copy DNA (cDNA). It is synthesized on RNA template with the help of reverse transcriptase enzyme and necessary nucleotides.

● Synthetic DNA (sDNA). It is synthesized with the help of DNA polymerases on DNA template or from free deoxyribonucleotides without a template.

Artificial synthesis of DNA on Template. In 1961, Kornberg and his coworkers synthesized DNA from a mixture of deoxyribonucleoside triphosphates, DNA polymerase enzyme, metal ions and a segment of viral DNA to act as a primer. The synthetic DNA showed biological activity. When introduced into bacteria, this DNA coded for the formation of virus particles similar to the one from which primer DNA was taken.

Artificial synthesis of DNA without a Template. Hargobind Khorana and his colleagues produced the first synthetic DNA in 1965 by purely chemical means. They synthesized the gene coding for yeast alanine-tRNA which needed only 77 base pairs. Unfortunately, this gene did not function. Khorana and his associates, in 1979, synthesized the gene coding for tyrosine – tRNA of E.coli. This gene had 207 nucleotide pairs. It functioned is the bacterium E.coli when introduced into it in combination with a phage DNA.

● Random DNA. It refers to small fragments formed by breaking a chromosome of an organism with the help of restriction endonucleases.

B. Procedure for the formation of Recombinant DNA.

It involves integration of passenger DNA to vehicle DNA. The DNA to be used as a passenger or vehicle is taken out from the cell by lysing it with a suitable enzyme. The DNA is isolated from other cell contents by ultracentrifugation and purified. Then both the passenger and the vehicle DNAs are cleaved by using the same restriction endonuclease so that they have complementary sticky ends. They are mixed under suitable conditions. Their complementary sticky ends pair and their ends are sealed with ligase. This produces a recombinant DNA.

2. Introduction of Recombinant DNA into Host. Three methods are used for introduction of recombinant DNA into the host: transformation, transduction and vectorless.

● Transformation. It is a process by which a cell takes up naked DNA fragments from the environment, incorporates it into its own chromosomal DNA, and finally expresses the trait controlled by the incoming DNA.

● Transduction. Transfer of DNA from one organism to another through a bacteriophage is called transduction.