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1. BEGR 424/Bio 324 Molecular Biology
William Terzaghi
Spring, 2013

2. BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William Terzaghi
Office: SLC 363
Office hours: MWF 10:00-12:00, or by appointment
Phone: [phone]
Email: [email protected]

3. BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William Terzaghi
Office: SLC 363
Office hours: MWF 10:00-12:00, or by appointment
Phone: [phone]
Email: [email protected]
Course webpage:
http://staffweb.wilkes.edu/william.terzaghi/BIO324.html

4. General considerations
What do you hope to learn?

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

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

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

8. General considerations
What do you hope to learn?
Graduate courses
1.learning about current literature
2.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
1.Pick a problem
2.Design some experiments

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

12. Plan A
1.Pick a problem
2.Design some experiments
3.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?
1.Bypassing Calvin cycle
2.Making vectors for Dr. Harms
3.Making vectors for Dr. Lucent
4.Cloning & sequencing antisense RNA
5.Studying ncRNA
6.Something else?

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

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

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

18. Plan B
Standard lecture course, except:
1.Last lectures will be chosen by you -> electives
2.Last 4 labs will be an independent research project
3.20% of grade will be “elective”
•Paper
•Talk
•Research proposal
•Poster
•Exam

19. Plan B schedule- Spring 2013
Date TOPIC
JAN14General Introduction
16Genome organization
18Cloning & libraries: why and how
21DNA fingerprinting
23DNA sequencing
25Genome projects
28Studying proteins
30Meiosis & recombination
FEB1 Recombination
4 Cell cycle
6 Mitosis
8 Exam 1
11DNA replication
13Transcription 1
15Transcription 2
18 Transcription 3

20. 20mRNA processing
22Post-transcriptional regulation
25Protein degradation
27Epigenetics
MAR1 Small RNA
4 Spring Recess
6 Spring Recess
8 Spring Recess
11RNomics
13Proteomics
15Exam 2
18Protein synthesis 1
20Protein synthesis 2
22Membrane structure/Protein targeting 1
25Protein targeting 2
27 Organelle genomes
29Easter
Apr1 Easter

21. APR3 Mitochondrial genomes and RNA editing
5 Nuclear:cytoplasmic genome interactions
8 Elective
10Elective
12Elective
15Elective
17Elective
19Elective
22Elective
24Elective
26Elective
29Exam 3
May1 ElectiveLast Class!
???Final examination

22. Lab Schedule
DateTOPIC
Jan16DNA extraction and analysis
23BLAST, etc, primer design
30PCR
Feb 6RNA extraction and analysis
13RT-PCR
20qRT-PCR
27cloning PCR fragments
Mar 6Spring Recess
13DNA sequencing
20Induced gene expression
27Northern analysis
Apr 3Independent project
10Independent project
17 Independent project
24Independent 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 10
6

Yeast has 2 x 10
7
Arabidopsis has 10
8
Rice has 5 x 10
8
Humans have 3 x 10
9
Soybeans have 3 x 10
9
Toads have 3 x 10
9
Salamanders have 8 x 10
10
Lilies have 10
11

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 ~10
9
bp

29. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~10
9
bp
mammals all have ~ 3 x 10
9
bp

30. Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~10
9
bp
mammals all have ~ 3 x 10
9
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 ~10
9
bp
mammals all have ~ 3 x 10
9
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!
Reason = variation in amounts of repetitive DNA

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

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

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

39. Cot curves
1. denature (melt) DNA by heating
2.Cool DNA

40. Cot curves
1. denature (melt) DNA by heating
2.Cool DNA: complementary strands find each other & anneal

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

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

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

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

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

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

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

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

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

50. Molecular cloning
To identify the types of DNA sequences found within each class they
must be cloned
Force host to make millions of copies of a specific sequence

51. Molecular cloning
To identify the types of DNA sequences found within each class they
must be cloned
Why?
To obtain enough copies of a specific sequence to work with!
typical genes are 1,000 bp cf haploid human genome is
3,000,000,000 bp
average gene is < 1/1,000,000 of total genome

52. Recombinant DNA
Arose from 2 key discoveries in the
1960's
1) Werner Arber: enzymes which cut
DNA at specific sites
called "restriction enzymes”
because restrict host range for
certain bacteriophage

53. Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA

54. Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA
methylase recognizes same sequence & protects it by methylating it
Restriction/modification systems

55. Recombinant DNA
Restriction enzymes create unpaired
"sticky ends” which anneal with any
complementary sequence

56. Recombinant DNA
Arose from 2 key discoveries in the 1960's
1) restriction enzymes
2) Weiss: DNA ligase
-> enzyme which glues
DNA strands together
seals "nicks" in DNA backbone

57. Molecular cloning
How?
1) introduce DNA sequence into a vector
•Cut both DNA & vector with restriction enzymes, anneal &
join with DNA ligase
•create a recombinant DNA molecule

58. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host

59. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
3) identify hosts which have taken
up your recombinant molecules

60. Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
3) identify hosts which have taken
up your recombinant molecules
4) Extract DNA