BIOL 2131 Lecture Notes - Lecture 8: Intron, Zygosity, Aneuploidy

• School: University of Windsor
• Department: Biological Sciences
• Course: BIOL 2131
• Professor: Hubberstey

Document Summary

Illumina!technology!doesn"t!give!you!your!genome!sequence,!nor!an!entire!fragment! of!dna,!rather!1!thing:!whether!aqt!or!gqc!base!pair!in!a!particular!snp: we!think!there!are!at!least!tens!of!millions!of!snp!throughout!our!genomes;!some!in, there!are!large!stretches!of!genome!that!are!identical!in!everyone!in!this!room;! genes!and!some!elsewhere! however,!there!are!other!regions,!like!in!snps!where!you!get!these!aqt!or!gqc! changes! A g genomic dna possibilities for each snp. In!the!second!case,!ag:: one!at!base!pair!from!mom,!one!gc!base!pair!from!dad, at!will!become!green,!whereas!gc!will!become!red! (the!technology!will!have, the!computer!will!convert!these!green!and!red!colors!to!yellow! (since!green! and!red!makes!yellow), aqt/cqg!heterozygote, both!red,!so!it!will!come!up!as!red!color, cqg!homozygote! 1. 2 million spots on each slide- each spot represents one snp. 23andme:: carrier!status:! of!them, family!inheritance:! mutations!in!a!gene, carrier!status:!40!diseases!that!are!known!to!be!caused!by!a!mutation!or, ex:!cystic!fibrosis, professor!has! variant!absent !for!the!40!diseases! !he!isn"t!a!carrier!for!any! !normal): you!isolate!mrna!from!different!tissues,!make!cdna,!add!fluorescently!labeled, afterwards,!look!at!expression!pattern, can!take!liver!cancer!cells!and!normal!cells!and!label!them!with!different!colors!in! nucleotides! nucleotides! order!to!try!to!see!what!is!being!expressed!in!cancer!cells!but!not!in!normal!cells! (or! vice!versa), hybridization!needs!to!work, differences!in!gene!expression!between!tissues!can!be!measured, this!gives!you!the!power!of!looking!at!the!entire!expression!profile!of!a!particular, see!figure!3. 24! tissue!at!a!particular!time! !makes!sense!because!most!genes!are!expressed!at!the: empty!black!spots:!genes!aren"t!expressed! (switch!not!turned!on!example), people!have!used!this!method!very!successfully!to!pull!out!genes!that!cause! same!level!in!2!different!cell!types! diseases, see!picture:!microarray!analysis!for!cancer!detection!and!treatment! !however,!there!are!other: a)!viral:!virus!vector!to!infect!bacteria!and!inject!dna, b)!liposomes:!dna!is!coated!with!lipid!membrane!and!dna/lipid!complex!fuses!with, very!efficient! plasma!membrane! (used!for!mammalian!cells), then!releases!dna!into!cell, very!effective, probably!most!widely!used!method!of!dna!transformation!for!mammalian, liposomes!don"t!work!for!bacteria,!yeast,!or!plants!due!to!them!having!cell! walls!which!block!liposomes!from!interacting!with!plasma!membrane! cells, c)!microinjection:!dna!is!injected!into!cells! (usually!fertilized!eggs), pretty!costly, d)!biolistics! (nanospheres):!dna!is!coated!on!a!bead!of!gold! or!tungsten!and!fired!into!cell!using!gene!gun! (plant!cells), called!a! dna!gun !!!analytical!ii!lecture!5!notes, used!just!for!plants!because!they!have!strong!cells! walls!which!require!a!lot!of!effort!to!get!dna! through!them, see!figure!3. 11!for!demonstrations!of!these! !has!to!be! be!single!stranded! single!stranded!in!order!to!get!hybridization: radioactivity, fluorescence, we!used!to!use!radioactivity!for!probes,!but!now!we!can!use!fluorescence!as!probes, nucleic!acid!is!usually!transferred!from!a!gel!to!a!solid!support! (filter),!probed,, or!probing!can!be!done!in!a!cell! (a!process!called!in!situ!hybridization), see!figure!3. 21!and!3. 12! southern!blotting!procedure ! washed!and!then!exposed! If!you!have!a!gene!that!you!want!to!identify!its!location!on!a!chromosome,!you!use! in!situ!hybridization: chromosome!has!to!be!denatured!with!a!chemical!in!order!to!unwind!it!and! break!the!hydrogen!bonds, add!single!stranded!denatured!fluorescently!labeled!dna! fluorescently!labeled!dna!probe, can!take!intact!cells!or!mitotic!chromosome!spreads!and!add!denatured, name!of!this!process:!fish! (fluorescent!in!situ!hybridization), old!technology!q!don"t!really!need!to!use!this!anymore!because!genomes!have!been, see!figure!3. 13! fish!technique ! sequenced!!this!is!the!power!of!genome!sequencing. ! chromosome!the!gene!is, tiny!spots!because!a!gene!on!a!genome!is!a!very!small!part!of!the!genome, you!can!see!the!chromosome!number!and!then!determine!where!on!the, can!do!this!in!interphase!or!in!the!mitotic!phase! Interphase can"t!use!hybridization!as!in!southern/northern!blotting: protein!detection!method, take!protein,!run!it!on!a!different!type!of!gel,!blot!it!to!a!solid!support,!but!then!you, you!have!to!use!a!compound!that!can!detect!a!particular!protein, thus,!we!use!antibodies!as!they"re!excellent!at!detecting!specific!individual!proteins, label!antibodies,!they!give!a!fluorescent!signal,!and!then!you!get!bands!on!a!gel, see!figure!3. 23! which!detects!protein!(analytical!ii!lecture!5!notes! detector)!involved!in!the!process, chemiluminescence, there!are!chemicals!and!luminescence! (visible!light!that!doesn"t!need!a!special, chemical!generation!of!light, because!we!produce!antibodies!and!get!them!from!companies,!they"re!not!labeled, antibodies!are!in!a!y!shape,!within!which!the!v!part!is!called!an! antibinding!region , companies!make!them!from!mice! Chemical generation of light: the!secondary!antibody!is!made!against!a!primary!antibody, thus,!you!have!protein!of!interest!!!primary!antibody!v!part!linking!to!it!, see!picture!of!these!attachments, see!picture!of!western!blot!example! secondary!antibody!v!part!linking!and!enzyme!at!the!end!