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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?
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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.


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QXHVWLRQ 5
In a recent publication Ze are told the folloZing:

B​RCA2​Ÿdeficie[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[ ​BRCA2​Ÿahh]ciajed ca[ceg​Â​​Ã​
BRCAÃ mai[jai[h ge[]me i[jeggijs jhg]kgh ijh fk[cji][ i[ jhe gedaig ]f DNA
d]kbleŸhjga[d bgeakh bs h]m]l]g]kh gec]mbi[aji][ ¥HR¦ ​Ä​​Å​​Æ​ I[ jhih dg]cehh jhe
gec]mbi[aji][ dg]jei[ RADÆ a[d BRCAÍ 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[ RADÆ 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[
HR​È​​É​ hkggehji[g jhaj addiji][al fk[cji][al d]mai[h i[ BRCAà c]kld jake ]peg CTD’h
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¦ŸPARP 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[ghŽ

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)?
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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).


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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?





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