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”.

EcoRIEcoRI 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: Jh5S0http:// 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).