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Lecture 44 Prof Duncan Shaw Recombinant DNA technology • First technical breakthrough in medical genetics was chromosome analysis in 1950s • Second is recombinant DNA, in the 1970s - 1990s • This will culminate in the complete DNA sequence of humans (the Human Genome Project) • There are now many methods available to analyse patients’ DNA in the lab, to identify mutations, to discover new genes, etc. How to purify a gene • First method is by “cloning”, i.e. introduce the gene into a bacterial cell then grow up large amounts and extract DNA (in vivo) • Second method is by “polymerase chain reaction” (PCR) using DNA polymerase to amplify the gene in a test-tube (in vitro) • Both methods have their uses but PCR is preferred in medical applications because it is quicker and cheaper Bacteria provide the means • Bacteria have been vital in developing DNA technology • Thermus aquaticus (which lives in hot springs) provides DNA polymerase enzyme for PCR • Escherichia coli (which lives in our guts) provides “plasmids” (mini-chromosomes) used in cloning • 100s of bacterial species provide “restriction enzymes” that cut DNA at specific sequences of bases (4 - 8 bases long) Applications • In biomedical research - to identify the genes responsible for human characteristics (including disease) • To analyse what goes wrong with these genes in disease (pathology) • To provide prenatal and presymptomatic diagnosis, carrier detection, risk calculation • New therapies (drugs, gene therapy) 95o 50o 72o 1 2 3 1.3kb 1.1kb Diagnosis by DNA of sickle-cell anaemia 1 = normal 2 = carrier (heterozygote) 3 = affected DNA sequencing by the Dideoxy (Sanger) method