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Molecular Genetics Techniques: Southern, Northern, Western Blotting, and More

This lecture review covers essential molecular genetics techniques, including Southern, Northern, and Western blotting, DNA sequencing, PCR (Polymerase Chain Reaction), and DNA microarrays. Learn how these methods analyze DNA and RNA, reveal gene expression patterns, and facilitate genetic research. The lecture provides insights into the mechanisms of each technique, including hybridization for blotting and enzymatic reactions for protein analysis. Gain a foothold in molecular biology by understanding the principles behind these powerful tools.

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Molecular Genetics Techniques: Southern, Northern, Western Blotting, and More

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  1. PBIO 427/527: Molecular GeneticsLecture 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 • 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

  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 • 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

  9. Characterization: DNA sequencing

  10. DNA sequencing

  11. Automated DNA sequencing Capillary electrophoresis

  12. 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

  13. The Polymerase Chain Reaction (PCR) • PCR is a cloning method without a host • Thermusaquaticus, 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 21-23 nucleotides each. The nuclease-RNA oligonucleotide complex binds and cleaves specific mRNA.

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