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Student No.: ____________________ QXHVWLRQ 3 In PLOS 2017 this paper Zas published ³A multiple[ PCR mini-barcode assa\ to identif\ processed shark products in the global trade´ Diego Cardexosa , AndreZ Fields, Debra Abercrombie, KeYin Feldheim, Stanle\ K. H. Shea, Demian D. Chapman It describes a method of determining Zhich shark makes up the meat offered at a market using an interesting piece of DNA, because . IQ KLgKeU eXNaU\RWeV, WKe 5S ULbRVRPaO DNA (5S UDNA) aUUa\ cRQVLVWV Rf PXOWLSOe cRSLeV Rf a KLgKO\ cRQVeUYed 120 baVe SaLUV (bS) cRdLQg VeTXeQce, VeSaUaWed fURP eacK RWKeU b\ a YaULabOe QRQ-WUaQVcULbed VSaceU (NTS) (LRQg aQd DaYLd, 1980) ( FLgXUe 1). TKe 5S UDNA UeSUeVeQWV a VXLWabOe caQdLdaWe fRU PCR-baVed geQeWLc VWXdLeV dXe WR VeYeUaO feaWXUeV: (L) Kead WR WaLO RUgaQL]aWLRQ Rf WKe 5S UDNA PXOWLgeQe faPLO\ PePbeUV; (LL) WKe NTS LV fOaQNed b\ WKe 5S URNA geQe cRSLeV LQ WKe 5S UDNA WaQdeP aUUa\, WKXV WKe PCR WecKQRORg\ caQ be XVed LQ WKe LVROaWLRQ Rf WKe NTSV; (LLL) WKe 5S URNA geQe LV KLgKO\ cRQVeUYed eYeQ aPRQg dLVWaQWO\ UeOaWed VSecLeV «; (LY) UeSeWLWLYe XQLWV Rf WKe 5S UDNA dR QRW e[ceed WKe OeQgWK Rf PCR aPSOLfLcaWLRQ UaQge« Bearing in mind the title of the paper, e[plain Zh\ this piece of code is of particular interest including: a) HoZ it Zill be tested (Zhat Zill be measured to tell the difference betZeen sharks, describe the test) b) HoZ just this region of the DNA Zill be prepared c) What Zould happen if the sample Zas not a shark at all? Page 8 of 19 Page(s) Student No.: ____________________ QXHVWLRQ 4 The Lac operon is shoZn in Wikipedia as the folloZing: There are tZo promoter regions Zith lacl in betZeen. What are the tZo promoter sequences doing? The MADSCI blog on this states that it is used in research Zith a T7 promoter. The\ use tZo sections of DNA, one Zith a T7 promoter and the gene of interest and the other Zith the T7 RNA pol\merase behind the lac operator. Which sequence is used in place of lacl in the diagram aboYe and Zh\? Using the information that ³because T7 is a Yer\ poZerful promoter, Yirtuall\ the Zhole cellular machiner\ becomes deYoted to the e[pression of \our protein´ to e[plain Zh\ the Lac Operon is used in this case particularl\ and hoZ this is likel\ to improYe the product. Page 10 of 19 Page(s) Student No.: ____________________ QXHVWLRQ 5 In a recent publication Ze are told the folloZing: BRCA2deficie[j jkm]kg cellh acckmklaje chg]m]h]mal ab[]gmalijieh a[d jhih ge[]me i[hjabilijs ih jh]kghj j] hape a cakhajipe g]le i[ BRCA2ahh]ciajed ca[cegÂÃ BRCAÃ mai[jai[h ge[]me i[jeggijs jhg]kgh ijh fk[cji][ i[ jhe gedaig ]f DNA d]kblehjga[d bgeakh bs h]m]l]g]kh gec]mbi[aji][ ¥HR¦ ÄÅÆ I[ jhih dg]cehh jhe gec]mbi[aji][ dg]jei[ RADÆÂ a[d BRCAÃ j]gejheg qijh ]jheg facj]gh degf]gm a geacji][ i[ qhich a damaged chg]majid daigh qijh jhe i[jacj hihjeg gehklji[g i[ jhe ercha[ge ]f ge[ejic i[f]gmaji][ fg]m jhe lajjeg j] jhe f]gmeg The DNA bi[di[g acjipijs ]f BRCAÃ ih i[jeggal j] ijh HR fk[cji][ ah ij facilijajeh jhe l]adi[g ]f jhe gec]mbi[aji][ dg]jei[ RADÆÂ aj DNA bgeakh Thih fk[cji][ ih e[hkged bs jhe DNA bi[di[g d]mai[ l]cajed aj jhe cagb]rs ¥C¦ jegmi[kh ]f jhe dg]jei[ ¥CTD¦ Ç Yej cellh gehihja[j j] DNA damage dep]id ]f jhe e[jige CTD ca[ hjill fk[cji][ i[ HRÈÉ hkggehji[g jhaj addiji][al fk[cji][al d]mai[h i[ BRCAÃ c]kld jake ]peg CTDh fk[cji][ T] jehj jhih hsd]jhehih qe khed dg]jei[ hec][dags hjgkcjkge dgedicji][ j]]lh ¥hee Skddleme[jags Mejh]dh ¦ a[d ide[jified a vi[c fi[geg ¥vf¦PARP like d]mai[ c][jai[i[g gehidkeh dgedicjed j] bi[d DNA i[ jhe ami[] ¥N¦ jegmi[kh ]f BRCAÃ jhaj age c][hegped i[ mammalh Oh ma[ jhaj h]k[dh c]mdlicajed S] I ][ls qa[j j] jhi[k ab]kj jhe fighj jhi[g BRCA ih h] [amed becakhe ij ih imdlicajed i[ bgeahj ca[ceg Acc]gdi[g j] jhih jhe []gmal ge[e ih d]i[g a g]]d jhi[g jhaj qe ki[d ]f k[]q h]mejhi[g ab]kj becakhe ij ih himilag j] CRISPR i[ h]me qash I jhi[k Erdlai[ jhe f]ll]qi[g jhi[gh Whs ih a d]kble hjga[d bgeak a hegi]kh dg]blem ¥defecjipe BRCA ih hjg][gls li[ked j] ca[ceg¦ Whaj ih jhe fk[cji][ ]f jhe hihjeg ¥a hec][d hjga[d ]f DNA¦ What is going on in this picture of ³Homologous Repair´ (borroZed from Wikipedia Author EmZ2012)? Page 12 of 19 Page(s) Student No.: ____________________ Page 13 of 19 Page(s) Student No.: ____________________ QXHVWLRQ 6 EPO is a protein that causes humans to make more red blood cells. It is an important Biotechnological ³drug´ for people Zho make too feZ blood cells on their oZn, such as the elderl\ and some serious illnesses. The artificial Yersion is produced b\ hamster cells using biotechnolog\ and is e[actl\ the same amino acid sequence as the human Yersion, but the side chains e.g. phosphate are different. EPO is also abused in sport, particularl\ in endurance eYents, including c\cling. To help preYent this tests haYe been deYeloped. It is described in a paper I found ³Testing for recombinant er\thropoietin Joris R. Delanghe, Mathieu Bollen, and Monique Beullens Cop\right Wile\ thus: «.the proteins in the concentrated urine samples are separated in a pH-gradient b\ IEF and the different Epo isoforms are Yisuali]ed b\ immunoblotting Zith monoclonal anti-Epo antibodies. Lasne [36] adapted the normal blotting procedure « to aYoid the nonspecific interaction of the used secondar\ antibod\ Zith urinar\ proteins. «.. The test can detect (natural EPO and different artificial Yersions). Page 15 of 19 Page(s) , Student No.: ____________________ In Fig. 1,a schematic image of the result obtained in a direct Epo test is represented. Fig 1 a) What is IEF (isoelectric focusing) and hoZ does it Zork? b) Wh\ should the phosphate side chains effect this? c) What is the point of using an antibod\? d) Wh\ not use tZo different antibodies? (Zhat is it about the molecule that the antibod\ recogni]es?) e) Wh\ are there seYeral lines for each protein? Page 16 of 19 Page(s)
