Recombinant DNA Techniques chapter 18 begins page 510 Part I

We’ll discuss a number of techniques and their applications. Restriction digest Southern Blot Northern Blot Western Blot Cloning

1. Restriction Digest: The Cutting of DNA Restriction Enzymes =

An example of cohesive or staggered cuts

Number of fragments vary between linear and circular DNA

How do you view the DNA fragments?

Bio 22 student DNA

EXAMPLES OF DNA AGAROSE GELS

An Application of Restriction Digest Chromosomal Disorders

Put your thinking cap on!

2 3 Wild type Hb Sickle cell Hb Restriction enzyme Restriction enzyme 1 band is too small to view here) 2 3 Restriction enzyme loss of cutting site

Wt= wildtype M= mutated M M Wt Wt Wt M

An Application to Forensic & Paternity Studies 2. The Southern Blot

1. Restriction Digest & gel electrophoresis TRANSFER DNA crime scene DNA suspect 2 DNA suspect 1 DNA victim

You must understand the reason for the “probe”!

But there are thousands of fragments!

So sometimes you have to really search for the differences between DNA.

Probe is complementary “hot” DNA

From: Restriction digest To result SUMMARY OF A SOUTHERN BLOT

Victim Suspect 1 Suspect 2 Crime scene Back to our crime scene

Techniques also are available to measure RNA and Protein 3. Northern Blot for RNA 4. Western Blot for Protein

Summary: Blotting techniques with probes Western Southern Northern

5. Technique: Cloning of specific genes or DNA fragments = restriction enzyme + = ligase

Cut vector Cut foreign DNA or gene Paste together

2 different antibiotic resistant genes Bacteria are now resistant to 2 antibiotics

Let’s apply these concepts to plasmid transformation.

Cut DNA here & insert foreign DNA

Will your transformed colonies with the insert be white or blue?

Summary of cloning DNA into a blue/white selection vector

End of part 1