begr 424/bio 324 molecular biology william terzaghi spring, 2013
TRANSCRIPT
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BEGR 424/Bio 324 Molecular BiologyWilliam TerzaghiSpring, 2013
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BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William TerzaghiOffice: SLC 363Office hours: MWF 10:00-12:00, or by appointmentPhone: (570) 408-4762Email: [email protected]
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BEGR424/BIO 324 - Resource and Policy Information
Instructor: Dr. William TerzaghiOffice: SLC 363Office hours: MWF 10:00-12:00, or by appointmentPhone: (570) 408-4762Email: [email protected]
Course webpage: http://staffweb.wilkes.edu/william.terzaghi/BIO324.html
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General considerations
What do you hope to learn?
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General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
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General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
• Learning how to give presentations
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General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
2. Learning current techniques
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General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
2. Learning current techniques
• Using them!
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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
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Plan A
1.Pick a problem2.Design some experiments
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Plan A
1.Pick a problem2.Design some experiments3.See where they lead us
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Plan A
1.Pick a problem2.Design some experiments3.See where they lead us
Grading?Combination of papers and presentations
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Plan AGrading?
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
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Plan ATopics?
1.Bypassing Calvin cycle2.Making vectors for Dr. Harms3.Making vectors for Dr. Lucent4.Cloning & sequencing antisense RNA5.Studying ncRNA6.Something else?
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Plan AAssignments?
1.identify a gene and design primers2.presentation on new sequencing tech3.designing a protocol to verify your clone4.presentations on gene regulation5.presentation on applying mol bio
Other work1.draft of report on cloning & sequencing2.poster for symposium3.final gene report4.draft of formal report 5.formal report
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Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives
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Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives2.Last 4 labs will be an independent research project
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Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives2.Last 4 labs will be an independent research project3.20% of grade will be “elective”• Paper• Talk• Research proposal• Poster• Exam
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Plan B schedule- Spring 2013Date TOPIC
JAN 14 General Introduction16 Genome organization18 Cloning & libraries: why and how 21 DNA fingerprinting23 DNA sequencing25 Genome projects28 Studying proteins 30 Meiosis & recombination
FEB 1 Recombination 4 Cell cycle6 Mitosis8 Exam 111 DNA replication13 Transcription 115 Transcription 218 Transcription 3
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20 mRNA processing22 Post-transcriptional regulation25 Protein degradation27 Epigenetics
MAR 1 Small RNA4 Spring Recess6 Spring Recess8 Spring Recess11 RNomics13 Proteomics15 Exam 218 Protein synthesis 120 Protein synthesis 222 Membrane structure/Protein targeting 125 Protein targeting 227 Organelle genomes29 Easter
Apr 1 Easter
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APR 3 Mitochondrial genomes and RNA editing5 Nuclear:cytoplasmic genome interactions8 Elective10 Elective12 Elective15 Elective17 Elective19 Elective22 Elective24 Elective26 Elective29 Exam 3
May 1 Elective Last Class!
??? Final examination
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Lab ScheduleDate TOPICJan 16 DNA extraction and analysis
23 BLAST, etc, primer design30 PCR
Feb 6 RNA extraction and analysis13 RT-PCR20 qRT-PCR27 cloning PCR fragments
Mar 6 Spring Recess13 DNA sequencing20 Induced gene expression27 Northern analysis
Apr 3 Independent project10 Independent project17 Independent project24 Independent project
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Genome Projects
Studying structure & function of genomes
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Genome Projects
Studying structure & function of genomes
• Sequence first
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Genome Projects
Studying structure & function of genomes
• Sequence first
• Then location and function of every part
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Genome Projects
How much DNA is there?
SV40 has 5000 base pairs
E. coli has 5 x 106
Yeast has 2 x 107
Arabidopsis has 108
Rice has 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
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Genome Projects
C-value paradox: DNA content/haploid genome varies widely
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Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
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Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp
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Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp
Other phyla are all over:
insects and amphibians vary 100 x
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Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
mammals all have ~ 3 x 109 bp
Other phyla are all over:
insects and amphibians vary 100 x
flowering plants vary 1000x
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C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
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C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
Arabidopsis has 10
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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
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C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA
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C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA
first demonstrated using Cot curves
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Cot curves
• denature (melt) DNA by heating
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Cot curves
• denature (melt) DNA by heating
dissociates into two single strands
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Cot curves
1. denature (melt) DNA by heating
2. Cool DNA
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Cot curves
1. denature (melt) DNA by heating
2. Cool DNA: complementary strands find each other & anneal
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Cot curves
1. denature (melt) DNA by heating
2. Cool DNA: complementary strands find each other & anneal
• hybridize
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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
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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]
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Cot curves
1) denature DNA
2) cool DNA
3) at intervals measure
[single-stranded DNA]
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Cot curves
viruses & bacteria show simple curves
Cot is inversely proportional to genome size
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Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
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Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”
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Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”Step 3 is ”unique"
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Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned
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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
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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
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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
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Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA
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Recombinant DNARestriction enzymes cut DNA at specific sitesbacterial” immune system”: destroy “non-self” DNAmethylase recognizes same sequence & protects it by methylating it Restriction/modification systems
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Recombinant DNA
Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence
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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
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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
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Molecular cloning How?1) create recombinant DNA2) transform recombinant molecules into suitable host
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Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
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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