L12 Molec Diag Flashcards
LO1: desc gen a numb of stand molec procs eg gene cloning, RA, DNA seq.
RE:
-bact prod endonucleases- recog and deg for DNA. Specif endonucleases (REs) cleave Pdiest bonds inside seq. Recog and cut specif RS. Mostly palindrome 4/5/6/8bp. Eg ecoli strain RY13 prods ecoRI recog said 5’GAATTC. Cuts after G=5’ and 3’ overhangs.
Prod sticky ends- can H bond each other as complem but not form Pdiest. Chance of find RS in bact 4^6. 100kB seq=20 ecoRI sites.
Bact protec own DNA by methyl.
-over 10000 bact screened for endonucleases. Over 2500 REs isol and purif. Over 200 diff RS recog. Eg ecoRI, BamHI, HindIII, PstI. Can get blunt ends. Use to isol frags of int btw sites. 600 commercially AV.
-why use RA- investig size DNA frags eg small delet/insert. Investig mut, often easier at DNA lev eg sickle cell. Investig DNA var eg DNA fingerprint for relat. Clone DNA.
LO1: cloning.
-take frag, make lot identic copies. RE isol DNA of int. To copy need out in vector DNA. DNA lipase= new Pdiest 2 exist pieces DNA. Dig at RS by RE= complem sticky ends. Align- mix and join with ligase. Staggered ends with diff RE can be complem and join eg BamHI and BglI- but RS NOT reformed.
-cloning- gene of int into vector eg plasmid.
Plasmid- small circ dsDNA. In bact. Extra chrom mini chroms. Carry genes to replic indep- indep ori’s, replic inserted gene. Can transf to other bact. Often carry AB resis genes= select for plasmid. Eg pUG19 LOT RSs, MCS=multip cloning site-conts RSs only present once in plasmid, allow insert foreign for amplif.
-gene cloning- DNA of int cut with RE, joins plasmid vec=recombin molec. Into to bact=transformation then select for cells cont recombin DNA eg AB resis ampicillin and grow them on it.
-4 basic steps:
Cut and join DNA of int. Use vac to carry DNA. Intro recomb DNA to host cells for replic. Id and isol clone cont DNA of int.
-why clone hum genes-
To make usef prots eg insulin. Used to isol and purif from non hum- diffic and foreign mater. Synth proinsulin by bact- proinsulin mRNA from panc (cont coding seq). Rev transc= proinsulin cDNA- join to plasmid= recombin DNA. Infect ecoli= transf bact prods proinsulin mRNA. Better to use mamm cells as have all necess prot transl modif sys to dev insulin- re chain and glycosylat and form S-S. Large scale hum insulin.
Find out what genes do- how/what exp/contr. Eg HTT.
Genet screen eg huntingtons, BRCA1/2, CF.
gene therap eg CF. multi dose clinic trials to re intro norm copies CFTR. Liposomes via inhaler for deliv to lungs. 39 pt PII trial. All impr and decr hosp comp to placebo. No AE due to adenovirus deliv sys. Also heart fail. Abn Ca rel from SR the re upt. SERCA2a down reg prev norm Ca proc. Give extra copies re est norm contrac.
LO2: desc theory behind DNA electrophor and how used to prov info about DNA frags.
-wells in agarose gel at anode. DNA tow cathode. Smallest frags trav fast so get further. Isol and vis frags. Sep frags on basis of size or shape.
Reqs- gel ma allow Sep. Buffer- salt etc, allow charge on DNA samps across gel. Pow supply gen charge diff across gel. Stain/detec to ID Sep DNA eg ethid bromide intervals btw bases-UV=Fluor.
LO3: expl PCR and appreciate its fundamental imp in genet testing.
-mullis 1983. TaqP disc 76-DNAP of thermos aquaticus bact hot spring.
Heat to 95 deg=donat. Break H bonds, melt low temp deps on GC: AT, more GC=higher as 3 H bonds. Add complem bits DNA bef cool, bind pref to complem seq in orig DNA=primers for DNAP=hybridisation. Then Cool to RT or 55 deg= renatures- primers bind. Use forw and Rev primer made to define reg cont DNA of int. Up to 72 deg for DNAP works 5’ to 3’ for each str. Rep 20-30x. 2^n molecs.
-PCR amplif targ DNA, often small samp. Thermos table DNAP, are others than Taq. Means don’t need add new enz as go as not donat. Thermocycler= temp cycles to denat, anneal, polymerise. Rep copying= exponen incr DNA.
-uses- amplif specif DNA frag. Investig 1bp mut eg Tay Sachs, SC. investig small delet/insert eg CF. investig var and genet relat eg DNA profiling and typing. V sensit. Diag CA/infec etc.
LO4: desc theory behind prot elecphor and how used to prov prot struc info.
-prot- charged molecs move tow anode or cathode in elec field. Sep by size, shape, charge. More ways than DNA.
Reqs- gel, buff maint charges, pow, stain/detec.
-thin vertic gel btw glass plates. Norm native (full folded) prot- Sep direc deps on intrinsic charge. Gen Miller travel faster through porous gel. Also dep on charge eg pos slower.
-serum prot elec- native eg
Dark bands= more prot. Often coomassie blue stain. Laser plots band intensity. Maj serum prot albumin. Lower peaks globulins (a, b, gamma). In monoclonal gammopathy- decr alb, incr gamma globins.
-sod dodecyl sulphate polyacrylamide gel elec- SDS PAGE.
More comm than native, uses unfold prot. Levs of prot exp and prot presence. Shape of folded affs movem through gel as does intrinsic charge of native. SDS Sep by MW only.
SDS= detergent= denat prot= no secondary or tertiary struc, just Lin pp chain. Break S-S by eg beta methanal. SDS also binds in fixed way to AA seq so same in all so just Sep by size. Prot now uniform neg charge. Move tow cathode. Unkn prot MW fnd by comp markers.
1SDS binds for ev 2aa. Bound SDS large neg charge.
-isoelec foc- Sep based on charge only. Col of gel- stable pH grad est after elec field applic. Pos=low pH. Prot sol added and elec field re applied. Prot mig til pH=PI=no net charge so stop.
-2D PAGE- IEF foll by SDS so Sep by charge and size. Good for complic samples. IEF gel on top of SDS gel, run SDS in diff direc=Sep prots with same PI. Imp in diag dis in diff tiss. Sep to sing types prot, comp intensity in dis vs normal show incr/decr exp. could be int/marker/useful for tx. Imp feats for proteomics.
-prot Id- proteomics- dig prot with trypsin. Perm mass spec- eg Id from 2D PAGE. Gen list pep sizes. Use database of predic pep sizes for known prots.
LO5: expl how Ab can be used in immunoassays and west blott to detec prot presence.
-specif cleavage of prots- less enzs than for DNA. Proteomics- anal all prots exp by genome. Molec diag= anal 1 purif prot.
-enz cleavage- trypsin cleaves after basic pos resids lys, Arg.staph protease cleaves after asp, glu. Chymotrypsin cleaves tyr, trp, Phe, leu. Endopeptidase lys-C cleaves lys.
-chem cleavage- cyanogen bromide- Met. Hydroxylamine cleaves Asn-Gly bonds.
-proteolysis Hb mutant- dig norm Hb with endo Arg-C cleaves after Arg=4 frag. Beta globin riverdale Bronx mut= extra Arg in N termin reg= 2 Sep bands= 5 tot bands.
-Ab- IgG struc- light and heavy chains. Var and constant domains. ABS. Bind specif Ag. Recog few AA on prot (epitope)-trigg imm resp.
-polyclonal Ab- prod main by B cells. Multip diff Abs. Specif to 1 Ag. Multip epitope- each Ab diff epitope. Infec Ag 3-4x at 2wk interval= Ab in blood, isol Ab to specif Ag.
-monoclonal- prod from 1 B cell. 1 identic Ab. Specif to 1 Ag. Recog 1 epitope. Ag to mouse, mouse spleen cells fused with cell cult myeloma cells= immort cell line-keeps div. selec and grow hybrid. Cells, selec cells making Ab of desired specif. Propag desired clones and grow in mass cult then isol Ab OR into mouse, induce tumour and isol Ab. Can freeze thaw.
-W blott to detec prot- eg detec exp in mixtures.
Transf SDS gel bands to sol ma eg nitrocellulose. Bind prim Ab against prot of int. Bind secondary Ab against prim. Secondary us enz or Fluor linked. Immunoblot.
-enz linked immunoabsorbent assay (ELISA)- meas conc in sol.
Indirec ELISA- Ag coated well. Wash, specif Ab binds Ag. Wash, enz linked Ab binds specif Ab. Wash, subst add and conv by enz to col prod- rate of col format propor to amnt specif Ab.
Work out how much Ab bind at a specif conc. Make stand curve of what levs Ag give partic resp eg insulin conc blood, plasma horms.
Radioimmunoassay use radiolab prim Ab.
Imp plasma horms- cortisol incr cushings decr addisons. RIA assay.
Insulin incr obesity, decr T1DM. RIA and ELISA.
TSH incr hypothyr decr hyperthyr. RIA and ELISA.
T3/4 incr hyperthyr decr hypothyr. RIA and ELISA.
LO6: underst basis for use enz assay.
-meas rate (prod over time) under defined conds, use to work out norm activ and comp abn.
-enz assays- mead prod:
Contin assay- spectrophotometry: light to monochromator t5o incid light to curettes with certain conc abs sp to transmitted light intensity to detec. Samp abs light of cert col. Chemoluminescence.
Discontin assay- radioactivity. Chromatog.
-meas of enz-
Metab disord- in tiss. Diag of dis- serum enz.
-clinic imp serum enzs- not norm in serum ,mark tiss dam. ALT+ST lot in liver- marker of liv dam/dis. Amylase/lipase- pancreatitis. Gamma-GT liv dam, incr by alc. ALP bone disord eg CA. Acid Pase- prostate CA. Plasma cholinesterase- decr in liv dis, inhib in organoP poisoning. CK-MI. LDH.
-enz elev after acute MI- serum marker.
Diff CK isoforms in diff types musc. BB brain. BM myocardium. MM skel/myocardium. CK isozymes neg mig tow anode. All incr in MI, MM mostly. Best MI diag cardiac troponin 1 (cTn1) by ELISA. Ab to recog, fast.
-meas metabol conc using enzs- monit [S] in serum/tiss.
Eg meas gluc conc with gluc oxidase. Catab:
Beta D gluc to D glucono-1-5-lactose. Test strips H2O2 conv to col dye. Gluc monit- gluc oxidase biosensor.
-enz assays perf at optim pH, temp, and ionic str. ions/cofactors prov. typ with high [S]. Meas prod/ disapp S. Diag metab disord when enz defic. Serum enz activ Indic dam.
Molec diag
- analyse DNA at gene lev- RE, DNA gel elec, PCR.
- analyse prot- prot elec, immunoassay, enz assay.
