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
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