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Recombinant DNA Techniqueschapter 18begins page 510 Part I 1. Restriction Digest: The Cutting of DNA How do you view the DNA fragments? An Application of Restriction DigestChromosomal Disorders An Application to Forensic Paternity Studies So sometimes you have to really search for the differences between DNA. Techniques also are available to measure RNA and Protein 5. Technique: Cloning of specific genes or DNA fragments Let’s apply these concepts to plasmid transformation. * * We’ll discuss a number of techniques and their applications. Restriction digest Southern Blot Northern Blot Western Blot Cloning Restriction Enzymes = An example of cohesive or staggered cuts Number of fragments vary between linear and circular DNA Bio 22 student DNA EXAMPLES OF DNA AGAROSE GELS Put your thinking cap on! Wild type Hb Sickle cell Hb Restriction enzyme Restriction enzyme loss of cutting site 1 band is too small to view here) 2 3 Wt Wt Wt M M M Wt= wildtype M= mutated 2. The Southern Blot 1. Restriction Digest gel electrophoresis TRANSFER DNA victim DNA suspect 1 DNA suspect 2 DNA crime scene You must understand the reason for the “probe”! But there are thousands of fragments! Probe is complementary “hot” DNA SUMMARY OF A SOUTHERN BLOT From: Restriction digest To result Victim Suspect 1 Suspect 2 Crime scene Back to our crime scene 3. Northern Blot for RNA 4. Western Blot for Protein Western Southern Northern Summary: Blotting techniques with probes + = restriction enzyme = ligase Cut vector Cut foreign DNA or gene Paste together 2 different antibiotic resistant genes Bacteria are now resistant to 2 antibiotics