Previously, it was very difficult to analyze DNA: DNA is enormously long and chemically "featureless" .
1970s brought new methods of DNA analysis; now DNA is the easiest of the cellular molecules to analyze.
Nucleotide sequences of several complete genomes have been determined. Human genome project is underway.The knowledge of the genome sequence will have a profound effect on the way deseases are diagnosed and treated.
Recombinant DNA and gene cloning are used to map mutations, to alter defective genes, produce medicinal compounds, and in many other applications.
A CLONE is a collection of cells that are all descendants of the same cell.
If a cell is placed on the surface of agar medium, it will start to divide and will eventually form a colony. All cells in the colony are descendants of one cell; thus, they represent a clone.
If the colony is inoculated in liquid medium and, say, a 10,000 L culture grown, all bacteria in the culture will be descendants of the same cell, and thus, they will represent a clone.
Gene cloning is producing a clone of organisms carrying an altered copy of a specific gene.
Gene cloning is usually used either to produce genetically altered organisms or to study genes.
The main steps of a gene cloning experiment are:
TOOLS FOR GENE CLONING include:
SCISSORS: RESTRICTION ENZYMES
GLUE: DNA LIGASE
SIEVE: GEL ELECTROPHORESIS
VEHICLE: PLASMID OR VIRAL VECTORS
RESTRICTION ENZYMES
RESTRICTION ENZYMES are sequence-specific nucleases.
Restriction enzymes recognize specific nucleotide sequences in DNA and cut both strands of the DNA molecule. Different restriction enzymes recognize different sequences in DNA. The recognition sequence can be 4, 5, 6, 7 or 8 nucleotides long.
Restriction enzymes cut DNA ONLY if the exact recognition sequence is present:
RESTRICTION ENZYMES often act as dimers; each restriction enzyme subunit recognizes THE SAME (5'->3') nucleotide sequence in complementary DNA strands:
As a result, restriction enzymes usually recognize palindromic sequences:
Some restriction enzymes, for example, Sma I, cut both DNA strands in the middle of the recognition sequence and produce "blunt-end" DNA fragments:
Many restriction nucleases produce staggered cuts in DNA which leave short single stranded "tails" at the ends of DNA fragments:
Single stranded tails produced in the result of DNA cleavage by restriction nucleases are known as cohesive or "sticky" ends. They can form complementary base pairs with the DNA ends produced by the same restriction enzyme.
Sticky ends of different DNA fragments produced by the same restriction enzyme can base-pair to each other. Linking together such fragments will produce a recombinant DNA molecule.
DNA LIGASE
DNA ligase is an enzyme that can connect two DNA fragments together.
Combining together fragments of different DNA molecules we can produce
recombinant DNA
