GENETICS GENE 3200
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This 4 page Class Notes was uploaded by Elna Nader on Saturday September 12, 2015. The Class Notes belongs to GENE 3200 at University of Georgia taught by Hall in Fall. Since its upload, it has received 19 views. For similar materials see /class/202263/gene-3200-university-of-georgia in Genetics (Graduate Group) at University of Georgia.
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Date Created: 09/12/15
Gene Exam 4 DNALibraries quot DNA 39 39 1 Compare the two types of DNA libraries 38 Genomic genomic DNA fragmented and ligated into cloning vector transformed into bacteria All regions of genome equally expressed cDNA mRNA isolated with oligoT primer cDNA using rev transcriptase cut mRNA with RNAse H use mRNA as primer and DNA pol to synth cDNA compliment methylase EcoRl sites in gene to protect add EcoRl linkers digest with EcoRI and clone into vector transform bact No promoter introns differs in different cells no regulatory regions 2 What is needed to express cDNA Why Have to directionally clone 5 to 3 in plasmid with promoter because promoter not in mRNA 3 Describe 5 ways to screen library Complimentation Have conditional mutants and transform with cDNA library any cDNA with the gene in it Screen 3 will compliment restrictive conditions and only those will grow S blot Use ssDNA probe to complimentary sequence Homology 3 Similar AA seq in another organism9look for conserved region reverse translation to make probes for libraryprimer PCR Transposon tag 4 Cross homozygous stable recessive mutation with transposon line F1 be hetero for mutation and transposon only if transposon move into gene area see recessive find location using sequence Positional cloning Use alleles and physical markers SNPs RFLPs VNTRs for recombination map to find region and then do chromosome walk in region 4 Describe S blotting technique 7 Spread clones on agar and grow colonies transfer to nylon filter treat to release denature and fix DNA on filter add ssDNA probe complimentary to desired area wash away excess expose to UV to see probe lD colony on plate and isolate 5 Describe positional cloning method 9 Find two markers flanking the gene cross two homozygous lines each differ in marker and in allele use recombination to make general map recomb based on what marker with what allele Use map region general location walk chrom into region by using end mol marker as probe for clones use ends clones for probe of nearby clones9continue till get overlapping clones contig9isolate clones in region and assay for phenotype to determine where gene is Reverse Genetics 1 Compare reverse and forward genetics Reverse identify gene mutate and id pheno how get pheno Forward id mut pheno gene and mut how mut cause pheno 2 Describe the two kinds of expression vectors for use in bacteria Bact promoter ShineDel Garno MCS transcription termination Euk promoterlike region TATA MCS polyadenylation transcription termination 3 What are three problems with bacteria expression vectors Can t control codon bias so may be underexpressed posttranslational modifications missed unless use euk r 39 vector 39 folding can only use cDNA 9 53 Iquot How was insulin produced in bacteria Describe method in general Make fusion gene with Bgal and fusion marker at Nterminal end gene and C terminal Bgal as cleavage site for CnBr fusion protein synthesis insert in MCS after prom gene and fus tag Describe the process of plant transformation Why work Use Agrobacterium which transfers TDNA from Ti plasmid into plants nucleus make disarmed plasmid with TDNA removed have conjugation ability and transformation vector with desired gene inserted between TDNA borders each has diff selectibility marker transfer into Agro and infect plant cell since totipotent one cell can grow into plant Describe the 2 processes of animal transformation 7 Inject into embryos or into gamete to form chimera Use posneg selection to get legitimate recombination heterozygote that is also homozygous for observe pheno insert into homozygous for opp pheno blastocyst F1 chimera hetero9mate F1 to wild type select F2 mut phenogerm cells hetero for mut cross the F29 F3 homo mut will either be homo normalheterohomozygous knockout How can you use viruses for transformation 6 parts replace Replace viral genome with IR for splicing viral package reg sequence cDNA and polyA IR What are two fates once transform Which preferred Why quot39 Uquot quot 39 39 bmal repair by NHEJ Legitimate gene with extensive homology only exchange in between homologous Prefer legit bc illegit could be in heterochrom incomplete insert or cause mutation Describe positivenegative selection Promote recombination using posneg selection insert pos marker in gene want to knockout gene form homologous region on ends and outside homolgous have negative marker transform into bacterial only those grow on plate lack neg marker have pos did homologous Describe 6 ways to inducescreen mutations Tilling Random mutate with EMS and use selection based on presence hetero DNA to J where mutant is SiteDirected mutagenesis Clone desired gene to be alter and denature add primer with mutantDna poldNTP9get mix doublestrands either primer or original degrade original using methylsensitive RE Sitespecific recombination CreloxP site Knockout library Insert exogenous DNA 9insertion mut knockout RNA interference silencing Introduce dsRNA comp to seq of gene9degrade mRNA Ectopic expression Fusion gene with expression for diff tissuetime Describe Crelox P use in animals flank transgene to remove with loxP and insert second transgene with Cre recombinase fusion pro with inducer domain cross ind with loxP to ind with Cre get hetero F19add inducer to get knockout mutation Describe tilling process Induce mutation with EMS to create M1 heterozygous mutation9propogate each into individual line collect DNA M1 and M29 PCR for gene of interest denature and reanneal the PCR products use endonuclease cleave single strand in heteroduplex indicate mut and original9run gel electrophoresis for each family and if see fragmentscut by endomutant9continue tilling for smaller and smaller size families 13 What are Zinc fingers and what two uses Similar Bind certain DNA sequences so if you make fusion protein with nuclease then will cause double strand breaks between two seq NHEJ and loss of function or if also add donor homologous to cut region will allow legitimate recombination Talen DNA 14 Describe 5 processes to measure expression of a gene Tell what show Name Show How Northern Blot Amt possibly tissue mRNA probe for electrophoresis shows difference in lengths and intensity shows amount W blot Amt Have membrane filter with protein detect with antibody and have secondary antibody with probe attach to antibody In situ hybridization Where Use I quot y DNARNA probe that glowcolor Antibody staining Where Use labeled antibody for celltissue as target Enhancer trap f enhancer Insert GFP in transposon and randomly insert if see expressed then insert bt enhancer and gene use seq to identify gene next to see how express Reverse Transcriptase Amt Reverse transcribe mRNA rev transcript and oligoT primer to PCR make DNA and then PCR amplification shows mRNA conc Gene Therapy 1 What is gene therapy and how done 3 Correcting genetic errors in living organisms inactivation improper gene new gene fight disease replace mutated 2 What are 4 challenges to gene therapy Choosing cells delivering gene have to correct in embryo to get all tissues but ethics wat to change and if worth risk 3 What make good target allele disease tissue 3 Recessive allele unregulated short gene Tissues if can be grown if rapid turnover transplant Disease more severe single organs no alternative treatment 4 Why is cystic fibrosis at first a poor candidate Why actually good Multiple alleles affected as are multiple organs pancrease repro lungs Found that its actually only a few alleles that affect the lung which is where can t treat and in the lungs only in airway ciliatedgoblet cells which can target with virus 5 How is CF used in gene therapy Problem Have defective virus that only makes capsid and add with CFTR gene virus w CFTR Lungs are designed to not take in foreign particles so prevent increase in expression Genomics 1 Describe the initial approach to human genome Clonebyclone construct physical map and clone genome into large piece use map find location then subclone into smaller pieces shotgun sequence rand cut and clone seq ends align sequence subclone9align subclone for clone align clone for genome 2 Describe the better approach to genome Straight to shotgun garment genome into small frage clone seq clone ends assemble 3 Why WGS better Problem How overcome No a priori knowledge so can do any org repetitive DNA prevent contig expand pearend seq H H H N H W P H U39I H 0 H l 9 5 N 0 Describe pairend sequence Drawback Gen seq from ends DNA frag of known size if seq flank repetitive than assign to same scaffold do pairedend read of libraries of various sizes and assign to scaffold Can t use for large rep elements that can t be clones telomere centromere Describe 4 steps in genome assembly Read give contig contig into scaffold scaffold by link map if no gaps scaffoldchrom Describe in bacteria 3 genomic library go to 140 contigs and thus 140 gaps Describe two types gaps and how fill Physical No clones available so probe genomic libraries for ends of scaffolds try and find missed or use PCR for ends of scaffold Sequence gap clones available so just use more clones Describe in Drosophila Why easier Do only euchromatin 3 genomic libraries form 850 scaffolds due to gene and phycial maps could locate Why is it so cheap now Fragment genome and sequence without clones What do once know sequence Two types Annotation to assing fxn Genome location of genefunctional sequence gene define fxn Describe the two annotation approaches and drawbacks Experimental cDNA compared to genomic sequence lack complete set cDNA clones alternative splicing not show fxn Computational use bioinformatics algorithms by ID open reading frames of 50 AA random stop codon every 20 AA so in large genome with large introns have worse prediction How D function of nonpro gene Have to sequence the RNA use experimental and comparative How find gene function Look for homolody assuming sim sequence is similar function use reverse genetics to confirm Describe gene families and how arise Genes evolutionary related from duplication events paralogof single gene of polyploid Describe 3 fates duplication Nonfunctionalization silencing accumulation of mutations in duplicate lose fxn Neofunctionalization mutation in duplication form new function Sub quot both duplicate and original mutate to have function put together is function original How are duplicates protected How determine freq Why Gene conversion homogenization prevents polyplodization prevents in multimeric pro prevent Examine loci with duplicates and id location to understand mechanism What notice about gene family euk obligate parasites Larger in larger genome Ob paras genome contaction Euk vary in gene density and number but all have more gene lower density than prok Describe ways new gene arise and lose 7 2 Arise exon shuffling neofunct reverse transcription derivation from transposase lateral gene transfer fusionfission de novo Lose accumulate mutation moderate deletion How tell gene from rev transcriptase What fate of most and how prevent Have polyA tail no intron smaller may not function because lack promoter Unfunctional bc no promoter insert next to transposon What is the C value paradox How explained Problem Amt DNA not based on complexity euk have larger repetitive gene number same paradox
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