1 / 33

PBIO 427/527: Molecular Genetics Lecture 2 - Review

PBIO 427/527: Molecular Genetics Lecture 2 - Review. Prokaryotic gene structure, processing & regulation Eukaryotic gene structure, processing & regulation Restriction enzymes & gel electrophoresis DNA cloning & cloning vectors Gene libraries & screening cDNA libraries & screening.

dewitt
Download Presentation

PBIO 427/527: Molecular Genetics Lecture 2 - Review

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. PBIO 427/527: Molecular GeneticsLecture 2 - Review Prokaryotic gene structure, processing & regulation Eukaryotic gene structure, processing & regulation Restriction enzymes & gel electrophoresis DNA cloning & cloning vectors Gene libraries & screening cDNA libraries & screening

  2. Prokaryotic gene expression

  3. Prokaryotic gene expression • Alternatively, see: • http://www.whfreeman.com/lodish4e/con_index.htm?99anm

  4. In prokaryotes, RNA polymerase binds to the -10 and -35 regions of the promoter relative to the start site of transcription (+1) promoter operator

  5. Eukaryotic gene organization enhancers silencers

  6. Eukaryotic gene organization and RNA processing

  7. Basic Transcriptional Mechanism and mRNA Splicing Animations • MCB Chapter 4-Basic Transcriptional Mechanism animation • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=04000&i=04010.01&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 • MCB Chapter 12-mRNA splicing animation • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=12000&i=12010.02&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=1211

  8. Eukaryotic gene expression

  9. MCB Chapter 4-Life Cycle of mRNA • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=04000&i=04010.02&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|&ns=0

  10. Recombinant DNA cloning procedure

  11. Recombinant DNA cloning procedure • See MCB Chapter 9 – Plasmid Cloning • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.05&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|&ns=437

  12. Restriction enzymes & DNA methylation

  13. Recognition sequences of some REs

  14. Mapping of restriction enzyme sites

  15. Cloning vectors and their insert capacities

  16. Plasmid cloning vectors • Three important features • Cloning site • Ori-an origin of replication • A selectable marker (ampr)

  17. pGEM-3Z

  18. Cloning foreign DNA into a plasmid vector Alkaline phosphatase-removes 5’ phosphate (P) groups of DNA molecules; BAP is more stable but less active than CIP T4 DNA ligase–joins 5’ phosphate (P) groups of DNA molecules to 3’ hydroxyl (OH) groups of DNA

  19. Some antibiotics commonly used as selective agents

  20. Genomic library construction

  21. Screening a genomic library using DNA hybridization to a (radio-)labeled DNA probeNote: a cDNA is commonly (radio-)labeled and used as a DNA probe to screen a genomic library

  22. Production of a (radio-)labeled DNA probe by the random primer method [uses the Klenow fragment of DNA polymerase] 5’ 3’ 5’ 3’ 3’ 5’

  23. The first step in making a cDNA library: Purification of polyadenylated mRNA using oligo(dT)-cellulose Note: selection of the proper source (organ, tissue) of the RNA is critical here!

  24. Complementary DNA or cDNA cloning:cDNA library constructionNote: ds cDNAs are typically placed in a cloning vector such as bacteriophage lambda (l) or a plasmid

  25. There are several possible ways to screen a cDNA library Using a DNA probe with a homologous sequence (e.g., a homologous cDNA or gene clone from a related species) Using an oligonucleotide probe based on a known amino acid sequence (requires purification of the protein and some peptide sequencing) Using an antibody against the protein of interest (note: this requires use of an expression vector) Plus/minus or differential screening (the least specific way)

  26. Screening a cDNA library using DNA hybridization to a (radio-)labeled DNA probe

  27. Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence

  28. Using polynucleotide kinase andg-32P-labeled ATP to radiolabel oligonucleotide probes

  29. Immunological screening of an expression cDNA library with a primary antibody and labeled secondary antibody; note the label is often an enzyme label like alkaline phosphatase or horseradish peroxidase, but it can also be 125INote: see also MCB Chapter 9 for a related animation http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.04&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=589

  30. Animations for two related uses of expression vectors • Expression cloning of receptor proteins-see MCB Chapter 9 • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=09000&i=09010.04&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=589 • Looking for protein-protein interactions with the yeast two hybrid system-see MCB Chapter 11 • http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?s=00010&n=11000&i=11010.01&v=category&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=798&t=&uid=0&rau=0

  31. Plus/min (+/-) or differential screening

  32. A cosmid cloning system:another possible cloning vector which can be used for genomic library but not for cDNA libraries

  33. In summary, you have seen: How to make and screen gene libraries How to make and screen cDNA libraries Several different cloning vectors including plasmids, bacteriophage lambda (l), and cosmids

More Related