Samary of molecular cloning .
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done by : asem shadid , college of medicine .
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Samary of molecular cloning .
- 1. • Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules (genes ) and to multiply their number in an organism. • Host organisms can be eukaryotic, bacterial or viral cells. • The purpose is to produce a large quantity of selected gene and it translation products. • Restriction enzyme are bacterial enzymes that cuts double-stranded DNA into smaller fragments. producing DNA segments called restriction fragments. DNA sequence that is recognized by a restriction enzyme is called a restriction site • This hybrid combination of two fragments is called a recombinant DNA molecule. • Recombinant DNA, with the plasmid containing the added DNA or gene has been formed. The recombinant plasmids are inserted into bacterial cells during a process known as transformation. • The introduced gene can begin producing its protein via transcription and translation. PROTEIN (Insulin) IS PRODUCED IN LARGE QUANTITY • POLYMERASE CHAIN REACTION • In a PCR experiment, two DNA primers of 20 base pairs are designed and synthesized chemically. We may want to detect if a person has a gene for diseases then the primer is synthesised with a short DNA sequence from this gene. PCR help to detect if a patient has this sequence in his genome • 3 reaction steps are performed in a PCR reaction. About 30 cycles • 1. Initial denaturation 2. annealing 3. extension Storage of DNA at - 4°C … Heating separates the double stranded DNA –Denaturation- Slow cooling anneals the two strands –Renaturation- Optimal temperature 72C • DNA is a giant anion in solution.
- 2. • Basic steps in DNA extraction • Break open cells and remove membrane lipids • Remove cellular and histone proteins bound to the DNA, by adding a protease, by precipitation with sodium or ammonium acetate, or by using a phenol/chloroform extraction step. • Precipitate DNA in cold ethanol or isopropanol, DNA is insoluble in alcohol and clings together, this step also removes salts. • Cutting DNA Fragments •Cohesive ends: fragments with short, single-stranded overhanging ends (Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.) •Blunt ends: even-length ends from both single strands •Gel electrophoresis-separation of DNA fragments by size through a gel medium -Smaller fragments migrate faster . Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge. •Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest .Is usually labeled for easy detection .Radioactive P32 or Fluorescent tag . •Microsatellites: variable number of copies of repeat sequences possessed by many organisms, which can be amplified by PCR . Combined with RFLP analysis to form more thorough fingerprint.