Understand the basic strategies for sequencing the genome of organisms – clone byclone vs shotgun. You should know the basic steps associated with each, theirsimilarities and differences.only 100-1000 we can only do short reads of DNA, must be broken into readablepiecesDNA is broken up into fragments cutting it with enzymes, or sheering DNA makingrandom breaks (shotgun), repeats make issues it is the unique sequences which allowus to order the fragmentsclone by clone: top down approach1. fragment genome into workable sizes2. clone fragments3. order fragments so they represent overlapping bits of chromosome( difficult and slow) done by restriction mapping and other methodssequence fragments that allow you to find out the linear sequence ofchromosomeslot of time figuring out the order of the clones, not necessary in shotgun methodorder first → sequence second → assemble (easy because we know order)shotgun sequencing:1. fragment genome into workable sizes2. clone fragments3. sequence all clonesa lot of redundancy in the collection of cloneslots of sequencing and sequenced data4. look at each fragment and try to link it to other fragmentsuse sequence similarity to join overlapping fragments(lots of fragments to assemble!)* sequence first → order/assembly second*most genome sequencing is done using shotgun because computers can do it * was used to first sequence human genome(too much repeat DNA make this process difficult)You should know and understand the basic steps in sequencing a genome –sequence,assemble, annotate4.**1. genome sequencing (clone by clone or shotgun)2. assemble genome3. annotate genomeKnow how genome assembly an annotation is done to the extent discussed in lecture.annotation: seeing that the functional sequences are- start by looking for known consensus sequences (TATA boxes, splice sites, openreading frames etc.)- Ab initio methods: predictions based on known “spelling and grammar”- functional annotation: sequence mRNA and compare it to the genome to figure outexpressed genes- comparative approach: use genes from other organisms to find genes in your speciesUnderstand and appreciate the power and uses of DNA hybridization. Understandhowit is used as the basis for asking question about the genome. This includes the conceptof a microarray. DNA hybridization: complementary sequences find each other- have DNA of known sequence can see if it binds to another unknown sequence tofigure out the sequence of the unknown DNAmicroarray: orderly arrangement of genome DNA fragmentsUnderstand that microarrays can be constructed to contain any number of ‘features’ ofa genome and then used to ask questions using those features.WHAT GENES ARE EXPRESSED?1. place DNA fragments in an ordered array2. collect all mRNA and convert it to cDNA, tag the cDNA fluorescent 3. apply tagged cDNA to microarray, complementary fragments will anneal4. genes that annealed to cDNA are expressed- used to compare genes under two different conditionsex. health/disease, time 1/time 2, treatment 1/treatment 2Expression studies with gene or open reading frame arrays, polymorphism studiesusing SNP arrays etc. Know how DNA hybridization is used in each case.Know what Single Nucleotide Polymorphisms are and how they can be used.(essentially just genetic markers)single nucleotide differences in sequencescompared with reference genome, our genome differs by 1 base in 1000SNP-chips to genotype people, microarray for detecting SNPsuse SNPs to find genes associated with phenotype or diseaseKnow what a Genome Wide Association Study is, how it is basically done and what thedata actually tell you. compare SNPs between 2 groups (disease vs. healthy) look at the differences to seewhat SNPs are related to the diseaseex. Rheumatoid ArthritisUnderstand and appreciate how genomic data are being used in the variety ofapplications discussed in class.metagenomics: sequencing DNA from environmental samplesdetermine what the genes aredetermine what the genes dohuman microbiome:- manipulating microbe communities can be beneficial to out healthKnow what cancer is and how it can arise. a disease of the cell cycle, failure of normal growth controls, tumorsall cancer results from somatic mutationscaused by virus or environmentKnow and understand the concepts of internal and external growth control signals andhow these relate to cancer.internal signals: cyclin/CDKexternal signals: hormones and growth factors membrane bound receptors can bind external factorsinner side of membrane receptors interact with kinases etc.Know what cyclins and CDKs are and their general role in the cell cycle. cyclins: intracellular proteins that vary in amount depending on cycle phase, functionas internal timers and triggerscyclins associated with cyclin dependent kinases--c-CDK phosphorylate specific proteins to regulate gene expressionUnderstand the regulation of G1-S transition as discussed in class (role of Rb etc.)RB = retinoblastoma proteinRB interacts with E2F (transcription factor for genes involved in proliferation)CDKs 4 6 2 phosphorylate RB in presence of D and Ephosphorylated RB dissociated from E2F → cell proliferationUnderstand the concept of external growth control signals and signaling cascades.RAS was an example of such a system. Understand that there are numerous suchsystems that depend on membrane receptors to detect particular growth signals.Theseform a network of signals that impinge on the nucleus. RAS signal transduction pathway involved in cell survival and deathRAS is one of a family of proteins, GTPasescan be activated by membrane receptors that respond to specific ligands, it thenactivates other proteins leading to cell growthAppreciate that the ‘network’ aspect of growth control relates to the multi-hit theory ofcancer mutagenesis.complex network of signaling pathways means that a defect will probably not be amajor problem because there are other control pathwaysUnderstand the basic evidence for cancer being viewed as a genetic disease. mutagens ~ carcinogens (implies that genetic damage is involved in cancer)chromosomal abnormalities correlated with cancerthere are some hereditary predispositions to cancers although this is rareKnow and appreciate the multi-hit theory of cancer. Know the concepts of driver and passenger mutations. driver: contribute to the development of cancer (cancer phenotype)passenger: mutations that are neutral with respect to cancer development but happento be in cells