PBIO 427/527: Molecular Genetics Lecture 3 - Review

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Presentation transcript:

PBIO 427/527: Molecular Genetics Lecture 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 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

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

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

Automated DNA 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

Microarray Result: Much analysis to follow

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 21-23 nucleotides each. The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.