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BIOL 301 - Midterm Summary notes (Terms, Experiments, Controls)

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McGill University
Biology (Sci)
BIOL 301
Nam Sung Moon

BIOL 301 Part 1: Analysing Gene Function Index: Lecture Contents Lecture 1: Scientific Method, Model Organisms, DNA extraction Lecture 2: Positional Cloning, Gene Mapping, SSLPs, PCR Lecture 3: Complementation, DNA cloning, transformation Lecture 4: Gene expression analysis, DNA chips, Northern Blot, RT-PCR Lecture 5: Promoter-reporter gene fusion, Gene expression analysis, Reverse Genetics, RNAi Definitions: Term Definition Forward Random Mutations, Select phenotype of Interest, find gene responsible. Genetics Identifying and isolating mutants that possess a phenotype of interest Reverse Identifying the function (phenotype) of a gene of interest. Genetics Common tools: Gene knockout and Over-expression. Gene Locating a gene of interest in the genome by identifying nearby landmarks by Mapping: determining linkage using recombination frequency. Closer genes are less likely to recombine. Phenotypical Associated with a visible Phenotype Marker Molecular Can visualize the phenotype by running marker DNA on a gel Marker Tools Tool Description Model Organisms Arabidopsis Thaliana – Plants Saccharomyces Cerevisieae – Baker’s Yeast, Buding Yeast Computer – Used for modeling, datamining. Gel DNA – Agarose Gel, smaller DNA fragment, higher % agarose. Fluo-DNA allows Electrophoresis to visualize under UV (Nucleic Acids) RNA, Oligonucleotides & Small PCR products – Agarose/Acrylamide or Acrylamide gel. Gel Polyacrylamide gels, SDS used to remove charge component (gives negative Electrophoresis charge) (Proteins) Gene mapping Used for positional cloning of a gene for which you have a mutant phenotype Cross homozygous mutant from F2 generation to a marker line Insertional Insert “tag” into a gene to disrupt it. Tail-PCR can be used to amplify flanking mutagenesis: sequences. Comparing to genome allows localization of gene. Tail-PCR: Use T-DNA primer and random oligos. The hope is that one oligo binds near your integration site to flank your tag and the surrounding sequence Allows you to identify the location of your tag. Chemical Ex: EMS. mutagens: Create random point mutations. Clone with positional or map-based cloning for localization. Genetic Look for mutant that fails to complement – Different Genes. Cross homozygous Complementation mutant with homozygous candidate. Test: Molecular Look for individual who complements – Your mutation is the same as test gene. complementation - Isolate (clone) mutant gene of interest DNA Test: - Insert SNA into mutant genome - Make sure it is expressed Extract cDNA Use reverse transcriptase to create DNA copy of mRNA Creating a PCR Amplify sequence with a primer containing an extra restriction site sequence product with restriction Sites Vectors Used to insert cloned gene into genome Retrovirus – Mammals T-DNA – Plants Transposon – Drosophila Use of a plant Use T-DNA flanked with LB & RB Vector Agro inserts region between LB & RB Vector Structure Make sure you have: - Species-Specific Vector - Insert - Endogenous Promoter (Can be tissue-specific, inducible or constitutive) Experiments: Linking a gene to a phenotype - Random mutagenesis - Identify a mutant with phenotype of interest - Clone the mutant - Confirm Mutation - Study gene’s Expression pattern Bioinformatics - Compare nucleotide sequence &predicted protein sequence with database of known sequence - Look for homology - May suggest a similar function Which gene is mutated in a mutant? Gene Mapping - Experiment: o Create a mapping population and identify homozygous mutant individuals from that population o Isolate DNA from these mutants o Perform PCR with primers specific to known polymorphisms (Amplify landmark region) o Run PCR on a gel o Identify RF% between mutant phenotype and molecular marker to determine map position of your gene o Perform further gene mapping experiments using markers located around the closest marker you’ve identified until RF=0 o Screen against a larger population size - Controls: o WT and homozygous mutant (Parental Strains) o Heterozygous mutant/WT (F1 generation) o DNA Ladder DNA Cloning Cloning With Restriction Enzymes: - Experiment: o Isolate DNA o Cut DNA with Restriction Enzymes (RE’s) in the presence of the appropriate buffer ▪ Phosphatase treatment prevents self-ligation ▪ Different RE’s prevents self-ligation ▪ Different RE’s allow you to orient inserts o Cut from: ▪ Cut previous cloned DNA out of another vector ▪ Cut genomic region out of BAC or YAC and purify the fragments. o Insert into the appropriate vector, digested with the same enzymes o Select for transformed vectors - Controls: o DNA ladder o No insert o DNA transformation controls Cloning DNA with PCR - Experiment: o Design primers using sequence information o Use PCR to amplify DNA o Cut from: ▪ From cloned DNA ▪ Directly from genomic DNA ▪ From cDNA clone/pool - PCR controls: o Sequence PCR-cloned DNA o No-Template control o Use as few cycles as possible After Cloning: - Insert into Vector - Transform Vector into E-Coli DNA Transformation: - Experiment: o Heat Shock or Electroporation o Grow bacteria in the presence of antibiotics for use as a marker o Select colonies/clones with insert o Infect Organism: ▪ Put gene into T-DNA ▪ Transform using organism-specific method - Controls for ligation & Transformation o Cut vector alone (control for contamination & self-ligation) o Uncut vector (test transformation efficiency) o Make sure gene is expressed (Can use RT-PCR or Northern Blot) o Make sure you have the right insert (Plasmid prep and Restriction Digests) o Multiple Transfections (Ensures phenotype is due to insert sequence, not insert location) Complementation Tests Molecular Complementation: - Experiment: o Create a mutant homozygous for gene X (Taken from F2 generation) o Clone WT gene X o Put insert (cloned gene) into vector specific to gene X species, do not forget to include promoter specific to organism into which you will transfect your gene o Put into Bacteria for cloning o Infect mutant with cloned gene o Phenotype rescue will occur if WT gene X is responsible for phenotype - Controls for ligation & Transformation o Vector only control o A construct expressing a mutant form o Multiple independent insertions ▪ Make sure rescue is due to sequence, not insertion location Genetic Complementation test: - Experiment: o Create a mutant homozygous for gene X (Taken from F2 generation) o Cross mutant with an individual homozygous for a known mutant copy o If complementation does not occur, your mutant and the known mutant are the same - Controls: o Sequencing of organism with your phenotype and of organism of known phenotype for confirmation Double-Check that you have the right insert
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