1. PBIO 427/527: Molecular Genetics Lecture 3 - Review
Southern, Northern & Western blotting
DNA sequencing
Polymerase chain reaction (PCR)
DNA microarrays
RNA interference (RNAi)

2. Characterization: RE mapping
Predict what would happen with a double digest.

3. Characterization: Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-developed by Edwin Southern

4. Characterization: Northern blot hybridization
X RNA X x
salt X
RNA
-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)
-reveals mRNA size (and approximate protein size), tissue- and organ-
specific expression, and kinetic patterns of expression

5. Characterization: Western blotting
X Protein
Enzyme
reaction or X
React with
Antibody X x
Buffer; requires electric current X
-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)
-requires the use of an electric current to facilitate transfer

6. Animations for Western Blotting
SDS gel electrophoresis- MCB Chapter 3
Western blotting or Immunoblotting- MCB Chapter 3

7. Characterization: DNA Sequencing
The dideoxynucleotide structure (note 3’H) is the key to dideoxy DNA sequencing

8. Dideoxy DNA Sequencing Animation- MCB Chapter 9

9. Characterization: DNA sequencing

10. DNA sequencing

11. Automated
DNA sequencing
Capillary electrophoresis

12. PCR Animation- MCB Chapter 9

13. The Polymerase Chain Reaction (PCR)
PCR is a cloning method without a host
Thermus aquaticus, a hot spring bacterium, produces Taq polymerase
Taq polymerase, unlike E. coli DNA polymerase, is not denatured at 95°C

14. Chemical synthesis of DNA (oligonucleotide synthesis using phosphoramidite chemistry)

15. DNA Microarrays:
An Introduction

16. Microarray
Result:
Much analysis to follow

17. RNA interference (RNAi)
dsRNA
sense
antisense
Binding of dsRNA-specific nuclease
Nuclease-ssRNA complex
Hybridizes to mRNA
cleavage
mRNA is cleaved!
A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of nucleotides each.
The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.