PBIO 427/527: Molecular GeneticsLecture 3 - Review

•Southern, Northern & Western blotting•DNA sequencing

•Polymerase chain reaction (PCR)•DNA microarrays

•RNA interference (RNAi)

Characterization: RE mapping

Predict what would happen with a double digest.

Characterization: Southern blot hybridization

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

Characterization: Northern blot hybridization

-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

X RNA

X

xsalt

XRNA

Characterization: Western blotting

-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)-requires the use of an electric current to facilitate transfer

X Protein

X

x Buffer; requires electric current

X

React withAntibody

X

Enzymereaction

or

Animations for Western Blotting

• SDS gel electrophoresis- MCB Chapter 3• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=03000&i=

03010.04&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=0

• Western blotting or Immunoblotting- MCB Chapter 3• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=03000&i=

03010.05&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=0

Characterization: DNA Sequencing

The dideoxynucleotide structure (note 3’H) is the key to dideoxy DNA sequencing

Dideoxy DNA Sequencing Animation- MCB Chapter 9

• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.06&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=592

Characterization: DNA sequencing

DNA sequencing

AutomatedDNA sequencing

Capillary electrophoresis

PCR Animation- MCB Chapter 9

• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.07&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=592

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

Chemical synthesis of DNA

(oligonucleotide synthesis using phosphoramidite

chemistry)

DNA Microarrays:An Introduction

MicroarrayResult:

Much analysis to follow

RNA interference (RNAi)

A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of 21-23 nucleotides each.A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of 21-23 nucleotides each.

The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.

dsRNA

Binding of dsRNA-specific nuclease

cleavage

mRNA is cleaved!

Nuclease-ssRNA complexHybridizes to mRNA

sense

antisense