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Download Resource and Policy Information Instructor: Dr. William Terzaghi
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BEGR 424 Molecular Biology William Terzaghi Spring, 2014 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: [email protected] 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: [email protected] Course webpage: http://staffweb.wilkes.edu/william.terzaghi/BEGR424.htm l 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 problem 2. Design some experiments Plan A 1. Pick a problem 2. Design some experiments 3. See where they lead us Plan A 1. Pick a problem 2. Design some experiments 3. See where they lead us Grading? Combination of papers and presentations 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 1. 2. 3. 4. 5. Plan A Topics? Cloning and analyzing oxalate decarboxylase and/or oxalate oxidase to see if they dissolve kidney stones in collaboration with Dr. VanWert Making vectors for Dr. Harms Cloning & sequencing antisense RNA Studying ncRNA Something else? 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 Plan B Standard lecture course, except: 1. Last lectures will be chosen by you -> electives 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 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 Date JAN 13 15 17 20 22 24 27 29 31 FEB 3 5 7 10 12 14 17 Plan B schedule- Spring 2014 TOPIC General Introduction Genome organization Cloning & libraries: why and how DNA fingerprinting DNA sequencing Genome projects Studying proteins Meiosis & recombination Recombination Cell cycle Mitosis Exam 1 DNA replication Transcription 1 Transcription 2 Transcription 3 19 21 24 26 28 MAR 3 5 7 10 12 14 17 19 21 24 26 28 31 mRNA processing Post-transcriptional regulation Protein degradation Epigenetics Small RNA Spring Recess Spring Recess Spring Recess RNomics Proteomics Exam 2 Protein synthesis 1 Protein synthesis 2 Membrane structure/Protein targeting 1 Protein targeting 2 Organelle genomes Mitochondrial genomes and RNA editing Nuclear:cytoplasmic genome interactions APR 2 4 7 9 11 14 16 18 21 23 25 28 30 ??? Elective Elective Elective Elective Elective Elective Elective Easter Easter Elective Elective Exam 3 Elective Final examination Last Class! Lab Schedule Date Jan TOPIC 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 4 Spring Recess 11 DNA sequencing 18 Induced gene expression 25 Northern analysis Apr 1 Independent project 8 Independent project 15 Independent project 22 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! 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 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 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