PHARMACOBIOTECHNOLOGY

TOOLS OF BIOTECHNOLOGY

Recombinant DNA (rDNA) is constructed outside the living cell using enzymes called “restriction enzymes” to cut DNA at specific sites and enzymes called “ ligases” to insert the cleaved piece of DNA (the “insert” ) into “ vector DNA” i.e., plasmid or viral DNA that will be able to enter a “host” cell or microorganism. Once the foreign DNA enters the host organism it is called “recombinant organism”.

RECOMBINANT DNA

Combining DNA from two different organisms

ENDONUCLEASES-type of enzyme in DNA strand.-produced nucleic acid strand breaks interior of nucleic acid strand.-restriction endonucleases-enzyme produced by bacteria that is used in recombinant DNA.-cuts open bacterial plasmid.(molecular

Scissors)-gene construct engineered to plasmid with ligases. Plasmids back to bacterium.

USEFUL PROPERTIES OF DNA

Restriction endonucleases can cut DNA at specific sites, leaving sticky ends for insertion of new DNA

Legend:The action of restriction enzymes

Restriction enzymes, also called restriction nucleases (EcoRI in this example) , surrounds the DNA molecule at the point it seeks(sequence GAATTC). It cuts one strand of the DNA double helix at one point and the second strand at a different, complementary point (between the G and the A base). The separated pieces have single stranded "sticky-ends," which allow the complementary pieces to combine.

The newly joined pieces are stabilized by DNA ligase.EcoRI, one of many restriction enzymes, is obtained from the bacteria Escherichia coli.

CLONING VECTORS

-carrier for DNA during the recombinant DNA process.-plasmid-piece of free-floating DNA in the cytoplasm of bacteria.-double-stranded, circular molecules that replicate independently of the chromosome.

A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose

SELECTION OF ALTERED CELLS

Antibiotic resistance gene used to identify recombinant cells

A palindromic number or numeral palindrome is a 'symmetrical' number like 16461, that remains the same when its digits are reversed. The term palindromic is derived from palindrome, which refers to a word like rotor that remains unchanged under reversal of its letters. The first palindromic numbers (in decimal) are:

0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 22, 33, 44, 55, 66, 77, 88, 99, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191 etc,

Most restriction enzymes leave sticky ends ,single strand extensions that will base pair with the opposite strand of another piece of DNA cleaved by the same enzyme , but some leave blunt ends.These annealed pieces will have single strand breaks (called “NICKS”) at the sites at which they were cleaved. Treatment of these mixed pieces with an enzyme called DNA LIGASE( usually T4 ligase from bacteriophage named T4) .

In gene cloning procedures, a gene corresponding to a specific protein will be joined with a cloning vector so that it can be transferred into a host cell. A cloning vector can be a plasmid or a bacteriophage. Plasmids are self-replicating, double stranded , circular DNA molecules that are usually maintained in the host cell independent extrachromosomal moiety.Plasmids will be characterized by size, host specificity (which organism it can exist in), and copy number, ex. The number of copies of a given plasmid usually found in a cell. Ones used for cloning purposes often have genetic elements such as antibiotic resistance genes that can be used to determine which cells have the plasmids.

Bacteriophages (viruses that infect bacteria ) are also used as cloning vectors, and foreign DNA inserted into the viral genome in an analogous manner. Viral DNA ( carrying as insert) get cells by viral infection, and the genes are expressed as the viral genome in expressed.

Hybridization probe

In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 bases long), which is used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single DNA strands and then hybridized to the target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane.Specific proteins can be detected by complimentary antibodies, leading to a color reaction, in a process called Western blotting.

Molecular DNA- or RNA-based probes are now routinely used in screening gene libraries.A LIBRARY is a collection of recombinant organisms, each of which contains a different fragment of DNA from the organism which naturally has the gene of interest in its chromosomes.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA.