Recombinant DNA Techniques chapter 18 Part I

techniques and their applications. 1. Restriction Digest (to be done in lab) 2.Southern Blot 3.Northern Blot 4.Western Blot 5.Cloning

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

EXAMPLES OF DNA AGAROSE GELS Ladder, marker or standard of known DNA sizes

An Application of Restriction Digest Chromosomal Disorders

Put your thinking cap on!

Wild type Hb Sickle cell Hb Restriction enzyme loss of cutting site 1 band is too small to view here) 23

Wt Wt M M Wt= wildtype M= mutated

An Application to Forensic & Paternity Studies 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!

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

Probe is complementary “hot” DNA

SUMMARY OF A SOUTHERN BLOT From: Restriction digest To result

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

Techniques also are available to measure RNA and Protein 3. Northern Blot for RNA4. Western Blot for Protein

Western Southern Northern Summary: Blotting techniques with probes

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