Recombinant DNA (DNA Cloning) SC.912.L.16.12

What is DNA cloning? When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a particular gene is copied (cloned)

Whole organisms are cloned too, but differently

DNA cloning and Recombinant DNA Major tool-restriction enzymes discovered in late 1960’s cuts DNA at restriction site highly specific manufactured by bacteria Cloning vector - carrier for moving DNA into a cell; such as a bacterial virus or plasmid into which foreign DNA can be inserted Recombinant DNA: joining together of two fragments of DNA that are not normally joined together (e.g. joining together of eukaryotic DNA and prokaryotic DNA - usually in a cloning vector)

Why Clone DNA? A particular gene can be isolated and its nucleotide sequence determined Control sequences of DNA can be identified & analyzed Protein/enzyme/RNA function can be investigated Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.

How is DNA cloned? DNA is extracted- here from blood Blood sample DNA is extracted- here from blood Restriction enzymes, e.g. EcoRI, HindIII, etc., cut the DNA into small pieces Different DNA pieces cut with the same enzyme can join, or recombine. DNA Restriction enzymes

Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”. Cut up “foreign” DNA that invades the cell. Type II and III restriction enzymes cleave DNA chains at selected sites. Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.

Basics of type II Restriction Enzymes No ATP requirement. Recognition sites in double stranded DNA have a 2-fold axis of symmetry – a “palindrome”. Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.

The action of a restriction enzyme, EcoRI Note: EcoRI gives a ‘sticky’ end

Cut and Paste DNA fragments with complementary ends (2 sticky ends)

DNA Cloning, II Bacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant”

DNA cloning, III The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids This insertion is called transformation

DNA Cloning IV The plasmids have naturally occurring genes for antibiotic resistance Bacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive

PCR polymerase chain reaction DNA doubled in each cycle starting with 1 molecule of DNA 1 million copies after 20 cycles Key to procedure is thermostable DNA polymerase (Taq polymerase) Ability to amplify minute amounts of DNA valuable in many disciplines basic and applied research criminal forensic science ecology analysis of ancient DNA