begr 424/bio 324 molecular biology william terzaghi spring, 2013
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BEGR 424/Bio 324 Molecular Biology William Terzaghi Spring, 2013. 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: (570) 408-4762 Email: [email protected]. - PowerPoint PPT PresentationTRANSCRIPT
BEGR 424/Bio 324 Molecular BiologyWilliam TerzaghiSpring, 2013
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]
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
General considerations
What do you hope to learn?
General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
• Learning how to give presentations
General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
2. Learning current techniques
General considerations
What do you hope to learn?
Graduate courses
1. learning about current literature
2. Learning current techniques
• Using them!
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
Plan A
1.Pick a problem2.Design some experiments
Plan A
1.Pick a problem2.Design some experiments3.See where they lead us
Plan A
1.Pick a problem2.Design some experiments3.See where they lead us
Grading?Combination of papers and presentations
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
Plan ATopics?
1.Bypassing Calvin cycle2.Making vectors for Dr. Harms3.Making vectors for Dr. Lucent4.Cloning & sequencing antisense RNA5.Studying ncRNA6.Something else?
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
Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives
Plan BStandard lecture course, except:1.Last lectures will be chosen by you -> electives2.Last 4 labs will be an independent research project
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
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
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
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
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
Genome Projects
Studying structure & function of genomes
Genome Projects
Studying structure & function of genomes
• Sequence first
Genome Projects
Studying structure & function of genomes
• Sequence first
• Then location and function of every part
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
Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Genome Projects
C-value paradox: DNA content/haploid genome varies widely
Some phyla show little variation:
birds all have ~109 bp
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
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
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
C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
C-value paradox
One cause = variations in chromosome numbers and ploidy
2C chromosome numbers vary widely
Haplopappus has 2
Arabidopsis has 10
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
C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA
C-value paradox
Chromosome numbers vary
So does chromosome size!
Reason = variation in amounts of repetitive DNA
first demonstrated using Cot curves
Cot curves
• denature (melt) DNA by heating
Cot curves
• denature (melt) DNA by heating
dissociates into two single strands
Cot curves
1. denature (melt) DNA by heating
2. Cool DNA
Cot curves
1. denature (melt) DNA by heating
2. Cool DNA: complementary strands find each other & anneal
Cot curves
1. denature (melt) DNA by heating
2. Cool DNA: complementary strands find each other & anneal
• hybridize
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
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]
Cot curves
1) denature DNA
2) cool DNA
3) at intervals measure
[single-stranded DNA]
Cot curves
viruses & bacteria show simple curves
Cot is inversely proportional to genome size
Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”
Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”Step 2 is intermediate: “moderately repetitive”Step 3 is ”unique"
Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned
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
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
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
Recombinant DNA
Restriction enzymes cut DNA at specific sites
bacterial” immune system”: destroy “non-self” DNA
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
Recombinant DNA
Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence
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
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
Molecular cloning How?1) create recombinant DNA2) transform recombinant molecules into suitable host
Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
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