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Molecular Biology Studies for Graduate Courses

An in-depth exploration of current literature and techniques in molecular biology. Focus on hands-on experiments and presentations. Choose between problem-solving approach or standard lectures. Assignments include gene identification and primer design.

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Molecular Biology Studies for Graduate Courses

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  1. BEGR 424 Molecular Biology William Terzaghi Spring, 2015

  2. BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointment Phone: (570) 408-4762 Email: terzaghi@wilkes.edu

  3. BEGR424- Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MWF 12:00-1:00, TR 1-2 or by appointment Phone: (570) 408-4762 Email: terzaghi@wilkes.edu Course webpage: http://staffweb.wilkes.edu/william.terzaghi/BEGR424.html

  4. General considerations What do you hope to learn?

  5. General considerations • What do you hope to learn? • Graduate courses • learning about current literature

  6. General considerations • What do you hope to learn? • Graduate courses • learning about current literature • Learning how to give presentations

  7. General considerations • What do you hope to learn? • Graduate courses • learning about current literature • Learning current techniques

  8. General considerations • What do you hope to learn? • Graduate courses • learning about current literature • Learning current techniques • Using them!

  9. Plan A • Provide a genuine experience in using cell and molecular biology to learn about a fundamental problem in biology. • Rather than following a set series of lectures, study a problem and see where it leads us. • Lectures & presentations will relate to current status • Some class time will be spent in lab & vice-versa • we may need to come in at other times as well

  10. Plan A Pick a problem Design some experiments

  11. Plan A Pick a problem Design some experiments See where they lead us

  12. Plan A Pick a problem Design some experiments See where they lead us Grading? Combination of papers and presentations

  13. Plan A • Grading? • Combination of papers and presentations • First presentation:10 points • Research presentation: 10 points • Final presentation: 15 points • Assignments: 5 points each • Poster: 10 points • Intermediate report 10 points • Final report: 30 points

  14. Plan A Topics? Making a probiotic strain of E.coli that destroys oxalate to help treat kidney stones in collaboration with Dr. Lucent and Dr. VanWert Making plants/algae that bypass Rubisco to fix CO2 Making vectors for Teresa Wasiluk’s project Making vectors for Dr. Harms Cloning & sequencing antisense RNA Studying ncRNA Revisiting blue-green algae that generate electricity Something else?

  15. Plan A Assignments? identify a gene and design primers presentation on new sequencing tech designing a protocol to verify your clone presentations on gene regulation presentation on applying mol bio Other work draft of report on cloning & sequencing poster for symposium final gene report draft of formal report formal report

  16. Plan B • Standard lecture course, except: • Last lectures will be chosen by you -> electives

  17. Plan B • Standard lecture course, except: • Last lectures will be chosen by you -> electives • Last 4 labs will be an independent research project

  18. Plan B • Standard lecture course, except: • Last lectures will be chosen by you -> electives • Last 4 labs will be an independent research project • 20% of grade will be “elective” • Paper • Talk • Research proposal • Poster • Exam

  19. Plan B schedule- Spring 2015 • Date TOPIC • JAN 12 General Introduction • 14 Genome organization • 16 Cloning & libraries: why and how • 19 DNA fingerprinting • 21 DNA sequencing • 23 Genome projects • 26 Studying proteins • 28 Meiosis & recombination • 30 Recombination • FEB 2 Cell cycle • 4 Mitosis • 6 Exam 1 • 9 DNA replication • 11 Transcription 1 • 13 Transcription 2 • 16 Transcription 3

  20. 18 mRNA processing 20 Post-transcriptional regulation 23 Protein degradation 25 Epigenetics 27 Small RNA MAR 2 Spring Recess 4 Spring Recess 6Spring Recess 9 RNomics 11 Proteomics 13 Exam 2 16 Protein synthesis 1 18Protein synthesis 2 20Membrane structure/Protein targeting 1 23 Protein targeting 2 25 Organelle genomes 27Mitochondrial genomes and RNA editing 30Nuclear:cytoplasmic genome interactions

  21. APR 1Elective 3 Easter 6 Easter 8 Elective 10 Elective 13 Elective 15 Elective 17 Elective 20 Elective 22 Elective 24 Elective 27Exam 3 29 Elective Last Class! ??? Final examination

  22. Lab Schedule • Date TOPIC • Jan 14 DNA extraction and analysis • 21 BLAST, etc, primer design • 28 PCR • Feb 4 RNA extraction and analysis • 11 RT-PCR • 18 qRT-PCR • 25 cloning PCR fragments • Mar 4Spring Recess • 11 DNA sequencing • 18 Induced gene expression • 25 Northern analysis • Apr1 Independent project • 8 Independent project • 15 Independent project • 22 Independent project

  23. Genome Projects Studying structure & function of genomes

  24. Genome Projects • Studying structure & function of genomes • Sequence first

  25. Genome Projects • Studying structure & function of genomes • Sequence first • Then location and function of every part

  26. Genome Projects • How much DNA is there? • SV40 has 5000 base pairs • E. coli has 5 x 106 • Yeast has 2 x 107 • Arabidopsishas 108 • Ricehas 5 x 108 • Humans have 3 x 109 • Soybeans have 3 x 109 • Toads have 3 x 109 • Salamanders have 8 x 1010 • Lilies have 1011

  27. Genome Projects • C-value paradox: DNA content/haploid genome varies widely

  28. Genome Projects • C-value paradox: DNA content/haploid genome varies widely • Some phyla show little variation: • birds all have ~109bp

  29. Genome Projects • C-value paradox: DNA content/haploid genome varies widely • Some phyla show little variation: • birds all have ~109bp • mammals all have ~ 3 x 109 bp

  30. Genome Projects • C-value paradox: DNA content/haploid genome varies widely • Some phyla show little variation: • birds all have ~109bp • mammals all have ~ 3 x 109 bp • Other phyla are all over: • insects and amphibians vary 100 x

  31. Genome Projects • C-value paradox: DNA content/haploid genome varies widely • Some phyla show little variation: • birds all have ~109bp • mammals all have ~ 3 x 109 bp • Other phyla are all over: • insects and amphibians vary 100 x • flowering plants vary 1000x

  32. C-value paradox • One cause = variations in chromosome numbers and ploidy • 2C chromosome numbers vary widely • Haplopappus has 2

  33. C-value paradox • One cause = variations in chromosome numbers and ploidy • 2C chromosome numbers vary widely • Haplopappus has 2 • Arabidopsis has 10

  34. C-value paradox • One cause = variations in chromosome numbers and ploidy • 2C chromosome numbers vary widely • Haplopappus has 2 • Arabidopsis has 10 • Rice has 24 • Humans have 46 • Tobacco (hexaploid) has 72 • Kiwifruit (octaploid) have 196

  35. C-value paradox Chromosome numbers vary So does chromosome size!

  36. C-value paradox Chromosome numbers vary So does chromosome size! Reason = variation in amounts of repetitive DNA

  37. C-value paradox Chromosome numbers vary So does chromosome size! Reason = variation in amounts of repetitive DNA first demonstrated using Cot curves

  38. Cot curves • denature (melt) DNA by heating

  39. Cot curves • denature (melt) DNA by heating • dissociates into two single strands

  40. Cot curves • denature (melt) DNA by heating • Cool DNA

  41. Cot curves • denature (melt) DNA by heating • Cool DNA:complementary strands find each other &anneal

  42. Cot curves • denature (melt) DNA by heating • Cool DNA:complementary strands find each other &anneal • hybridize

  43. Cot curves • denature (melt) DNA by heating • Cool DNA:complementary strands find each other &anneal • Hybridize: don't have to be the same strands

  44. Cot curves • denature (melt) DNA by heating • Cool DNA:complementary strands find each other &anneal • Hybridize: don't have to be the same strands • Rate depends on [complementary strands]

  45. Cot curves • 1) denature DNA • 2) cool DNA • 3) at intervals measure • [single-stranded DNA]

  46. Cot curves viruses & bacteria show simple curves Cot is inversely proportional to genome size

  47. Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive”

  48. Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive” Step 2 is intermediate: “moderately repetitive”

  49. Cot curves eucaryotes show 3 step curves Step 1 renatures rapidly: “highly repetitive” Step 2 is intermediate: “moderately repetitive” Step 3 is ”unique"

  50. Molecular cloning To identify the types of DNA sequences found within each class they must be cloned

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