Recombinant DNA Techniques Playing god for fun and profit! Or What happens when a mummy test tube and a daddy test tube love each other very much. What is Recombinant DNA? • DNA from one organism that has had DNA from another organism inserted in it. It may be from a different species. Tools • Really it is just cut and paste job using chemicals instead of scissors and glue. • The scissors are Restriction enzymes. • The glue is called DNA Ligase. The Scissors: Restriction Enzymes • Restriction Enzymes are enzymes that cut DNA at a specific sequences of bases. • Some will make a straight cut so the cut ends of the DNA are even in length. • Others will make a jagged cut with one strand longer than the other; this is known as a “sticky end”. EcoRI digestion produces "sticky" ends, whereas SmaI restriction enzyme cleavage produces "blunt" ends The Glue: DNA Ligase • DNA ligase is used to glue the cut ends of DNA together. • Works with both sticky and blunt ends but sticky ends produce a better yield. • Why would you use blunt ends? Let’s see the cutting and pasting process in full: • http://www.youtube.com/watch?v=aA5fyW Jh5S0 Vectors • Once you have created your recombinant DNA you then have to find a way to stick it inside your organism. • How you do this will depend on: – What organism you want to put it in. – Whether you want the genetic change to be permanent – If you want the change to be pass on the offspring – How precisely you want to place the DNA in the genome Bacteria • Plasmids – Bacteria naturally have small circles of DNA with genes on them (these is different from their main chromosomal DNA) that they may pass between themselves. – Recombinant plasmids, as seen in a previous slide can be put into bacteria to give them new genes. – They may be inserted into bacteria using various methods. Eukaryotes • Viral vectors • Yeast artificial chromosomes. • Gene guns Identifying Cells with Recombinant DNA • In bacteria: – add antibiotics resistance genes to the recombinant plasmid. – add a marker gene that causes a colour change. • Use antibodies to detect protein of inserted gene. • Complementary RNA or DNA probes (these are probes that have DNA that is complementary to your inserted genes that have a marker (e.g. florescent dye) on them).
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