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INTRODUCTION TO THE WAKSMAN RESEARCH PROJECT DNA Sequence Analysis of the Duckweed Wolffia arrhiza PROBLEM What genes are present in Wolffia arrhiza? What are the functions of these genes? WHY DO WE WANT TO IDENTIFY GENES AND THEIR FUNCTIONS? Identifying genes and their functions we can cure: Genetic disorders abnormal genes making abnormal products-block abnormal genes not making essential products-replace Cancer Identify genes involved-turn on or off Ongogenes-turn off Tumor suppressor-genes-turn on Identifying genes and their functions we can cure: Diabetes Add insulin making gene Spinal cord injuries Turn on nerve cell genes for cell division Infectious diseases Turn off vital genes-kill organism Alzheimer's Turn off genes making abnormal proteins Why Wolffia arrhiza? Smallest flowering plant Grows in slow moving fresh water -world wide Fast reproduction - doubles in a few days QuickTime™ and a decompressor are needed to see this picture. QuickTime™ and a decompressor are needed to see this picture. Duckweed-The little plant that can save the world Potential biofuel source • Under cold temperatures can accumulate 40%-70% starch • Sink to bottom of ponds • Starch can easily be converted to sugar for fermentation • Will not compete with food crop production Bioremediation • Grows in contaminated (polluted) water • Sequesters or degrades contaminates such as lead, arsenate, halogenated compounds • Reduces excess nitrogen and phosphate from waste water Potential food source Possible source of inexpensive protein Reduces global warming and produces oxygen Duckweed may serve as a model organism Model Organisms: A model organism is a species that is extensively studied to understand biological phenomena QuickTime™ and a decompres sor are needed to s ee this picture. Model Organisms: It is understood that discoveries made in model organism will provide insight into the working of other organisms Quic kTime™ and a dec ompres sor are needed to see this pic ture. Quic kTime™ and a dec ompres sor are needed to see this pic ture. QuickTime™ and a decompressor are needed to see this picture. Model Organisms Model organisms are widely used to explore potential causes and treatments for human diseases when human experimentation is unfeasible Why model organisms work? Model organism strategy made possible by the common descent of all living organisms and the conservation of metabolic and developmental pathways and genetic material QuickTime™ and a decompressor are needed to see this pictur e. Why are we able to apply knowledge obtained from model organisms to humans? Evolution-similarities among organisms are based on common ancestries Universal genetic code All use same four nucleotide bases All use same 20 amino acids All use ATP All made up of cells Important genes are conserved genes The more essential a gene is the less likely is to have mutated. Thus essential genes will be very similar among organisms In order to study DNA, it must be amplified and eventually purified and stored To purify a gene means to isolate gene from rest of DNA and cell For many years, biochemists had tried to purify genes. But they were frustrated because they are hard to purify. Because genes are composed of A’s, C’s, G’s, and T’s, they all pretty much are chemically alike. Also genes are parts of chromosomes. Chromosomes break easily and randomly, often in the middle of genes. So how did scientists eventually purify individual genes? Amplification means to make many copies of a gene Once you have many copies of a purified gene you need a way to store it for future use and research How can you accomplish purification, amplification and storage of a gene? VECTORS ALLOW US TO ACCOMPLISH ALL THREE TASKS What is a vector? Any vehicle that can carry DNA into a host cell Once inside the host cell it has the ability to replicate itself and any inserted DNA Amplification can be accomplished thru cloning with a vector What are the three steps in cloning? DNA of organism must be broken down into smaller pieces Pieces of DNA must be joined to another piece of DNA (vector) that can replicate itself and the DNA of interest Vector plus its joined insert must be introduced into a living cell (living cells act as copying machines) Many types of vectors YACS-400,000 bp BACS-100-300 bp Lambda phage-20,000 bp Cosmids-40,000 bp Plasmid-type of vector we are using What is a plasmid? Type of vector Small, circular, self replicating extra piece of DNA completely distinct from chromosomal DNA Found naturally in bacteria and yeast Contains a small number of genes not required for survival under normal conditions What is a Plasmid? Can give a survival advantage to bacteria living in a stressed environment Example-antibiotic resistance Due to high energy requirement of maintaining and replicating plasmids, only plasmids that confer an advantage are kept by organism What is a Plasmid? Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. Some plasmids are present in E. coli at 200-500 copies/cell What is a plasmid? QuickTime™ and a decompressor are needed to see this picture. Two Types of plasmids QuickTime™ and a decompressor are needed to see this picture. What type of plasmid are we using? What are important properties of pDNR-Lib? Important feature of plasmid Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Plasmid Characteristics Ori Selectable Marker 3.6 Kb Color screen (not in this plasmid) MCS Circular DNA Color Screen-not found in pDNR-LIB but important in many other types of plasmids What tools do we use to cut DNA of interest and join it to a plasmid? QuickTime™ and a decompressor are needed to see this picture. Must get plasmid with insert into host cell Transformationintroduction of foreign plasmid into a bacteria What are restriction enzymes? Cut DNA at defined sequences 4-8 bp long called restriction sites Cut phosphodiester bonds that link nucleotides together Cut in a precise and predictable manor, thus reproducible Restriction fragments-piece of cut DNA Where do restriction enzymes come from? Example-EcoR1 restriction enzyme QuickTime™ and a decompressor are needed to see this picture. How do we know their will be our restriction site Restriction site sequences occur randomly many times in a long DNA molecule Probability of six base sequence 46=4,096 bp How are restriction enzymes named? EcoRI from Escherichia coli BamHI from Bacillus amyloliqueraciens PvuI and PvuII are different enzymes from same strain. Genus-species-strain-order of discovery What happens if we cut Duckweed DNA and our plasmid with EcoR1? Example on board Why do we add ligase? Link together nucleotides Phosphodiester bonds Dehydration synthesis When we work with enzymes must create optimal working environment Need buffer (pH, salt conc) Proper temperature Poor conditions may: deactivate enzyme cause starr activity What restriction enzymes do we use in our research? Sfi used to cut Duckweed DNA and plasmid for joining Cloning W.a. cDNA fragments into the pDNR-Lib polylinker A.f.Insert insert Ava1-cuts insert out of plasmid AvaI CPyCGPuG Py stands for pyrimidine- T or C Pu stands for purine - A or G CTCGAG CTCGGG CCCGAG CCCGGG Information on Restriction Enzymes Serve as landmarks in plasmid to help find insert SMA I ECORI XBA I XHO I HIND III CCCGGG GAATTC TCTAGA CTCGAG AAGCTT What will be our first step? We will start with a DIGEST Cutting our insert out of the pDNR-Lib plasmid What is most important to remember? Always keep enzymes on ice (denaturation) Always use fresh tips Keep record in log book clone name date of digest How do we name our clones? 13ME01.09 13=PHHS ME=Initial of person who made clone 01=Number assigned to clone 09=Year of project What materials should I have for Digest? Ice in bucket Miniprep DNA AVA1 enzyme 10X buffer ddH2O 10X loading gel Microfuge tubes Incubator 37C Pipetman Pipet Tips Sharpie Microcentrifuge Tube holder Lets practice pipeting QuickTime™ and a decompressor are needed to see this picture. DIGEST PROCEDURE Label I microfuge tube 5X Digest mix Label ____ tubes with clone name and digest DIGEST PROCEDURE 1 Reaction mix dd H2O 7ul 10X buffer 2ul Miniprep DNA 10ul AVA1 1ul 5 reaction mix dd H2O 35ul 10X buffer 10ul Miniprep DNA * Ava1 5ul DIGEST PROCEDURE Mix reaction mix by pipeting up and down Add 10 ul of reaction mix to each microfuge tube labeled with a clone name Add 10ul of the corresponding DNA to the corresponding labeled tube Mix each tube by tapping or in centrifuge at low for a few seconds Incubate for 1hour at 37C Add 2ul of 10X loading gel Store in freezer -20C Next Procedure performed on miniprep DNA is PCR What is PCR? How does PCR work? Denatures DNA Primers anneal Taq polymerase extends primers Repeat cycle 30 times Performed in a thermocycler SHOW ANIMATIONS 95C 50C 72C What materials are needed to perform PCR? Thermocycler ddH2O Primer forward Primer Reverse Miniprep DNA Pipetman Pipet tips Vortex Microfuge tubes Ice with bucket Rack for microfuge tube Rack for PCR Tube PCR tube with bead Bead contains (taq polymerase, buffer, and nucleotides) PCR PROCEDURE Label four tubes 50 fold dilution and clone name Label PCR tubes with clone name Label 1 tube 5RX mix PCR DILUTE DNA FOR PCR To each tube labeled 50 fold dilution add: 98ul of ddH2O 2ul corresponding miniprep DNA Mix by vortexing WHY DILUTE PCR PROCEEDURE 1 REACTION MIX ddH2O 18ul Forward Primer 2.5ul Reverse Primer 2.5ul DNA (diluted) 2ul 5 REACTION MIX ddH2O 90ul Forward Primer 12.5ul Reverse Primer 12.5ul DNA * PCR PROCEDURE Mix reaction mix by vortexing Add 23ul of reaction mix to each labeled PCR tube Add 2ul of diluted DNA to appropriate PCR tube Mix by gentle tapping Place in thermocycler Record location in thermocycler
