L13 Molec Techs And Diag Flashcards
LO2: expl imp of PCR in genet testing.
LO3: desc DNA hub and appreciate role in event testing. Hyp using probe. Radio/Fluor complem ss lab DNA renat= heteroduplex. Blott/Fluor.
LO4: underst how PCR, RA, DNA hyb can be used in allele specif tests.
LO5: disc techs anal DNA at gene and chrom lev.
-PCR specif primer. Can make complem match allele seq. Mism at 5’= bind less tight but make prod. At 3’=taqP can’t read templ and ext. so test for sing base mut.
-S blott hyb- allele specif probe. Hyb to Sep DNA. Mism= bind less tight. Gel elec to mem, vis specific frags. DNA gel to sol=blott. Hyb= 2 ss complem DNA seqs reform H bonds=dsDNA frag. S blott radio/Fluor tag. N blott if RNA.
-ANAL GENE EXP-
N hyb- anal RNA. On gel. RNA degs more. See size and quant.
W blott prot.
RT-PCR- mat mRNA present when genes exp. use T primer the RT uses RNA as templ, makes ss cDNA copy, make primers for cDNA=a amplif dsDNA.
LO3: microarray
-blott need know gene looking for- few probes. Microarray look at lot genes or if unkn which gene-GWA. 1000s genes 1 chip. Array of DNA dots by pin.
-mRNA isol from samp (norm vs dis eg CA). RT lab red vs green Fluor= comb targs and hyb to microarray. Ev dot=diff clone. Anal 10000s genes simult. See condition exp of mRNA btw 2 conds. Both samp exp same gene=brown. Many shades= specif-amnt exp. comp backg too. Often swap cols and rep.
-array comparat genome hyb- can have duplic/delet etc.
Someth wrong, don’t know where. DNA from pt and control. Lab diff fluorochromes then IX eq quants and hyb to array of clones. Work out red:ratio of cell, align database of clones. Nothing wrong=brown. If delet in pt then only Fluor contr col. Duplic= Fluor more tow pt col.
-why use array- investig 1000s genes simult. Investig chrom delet/duplic- only if micro as can see macro. Investig conditional gene exp.
LO3: DNA fingerprinting
-Jeffrey’s 1984. Now DNA profiling.
Genome has lot rep DNA seqs-microsatellite- show copy no var. mat and pat copy. Cert areas v var. siblings sim.
-look at 1 var reg- each parent 2 diff size bands As diff no’s reps. Kids one mat and one pat band. Show relats.
Ev lane=indiv. Can look at multip var regs.
Eg disputed son- ev band in either mum or sibling- build up dad.
-DNA prof uses smaller reps to fingerprint= small rand reps (STR)- v var. PCR amplif one var reg. 2 binds per var reg. Less than one var reg= prof.
-why use DNA profiling- forensics- even tiny samp. Paternity dispute.
ANALYSE DNA AT CHROM LEV
- stain=band patt. Investig chroms us need dividing mitot cells. Cells collec from tiss samp eg blood, BM, feral, amn fluid, chorionic villus, and cult in vitro. Lymphoc must be cult in phytohaemagglutinin to induce cell div. cells then arrested when most in late prophase/early metaph- chrom v cond and replicated but not organised on metaph plate. Metaph spreads (metaph chroms spread out) can be anal by staining proceds incl G banding and FISH.
- chrom anal- karyotyping- banding chrom specif. Stain show transloc etc. Also size and no of chroms. Karyot is pic of full set stained metaph chroms organised acc to no.
- FISH- hyb. Need know what look for, make probe to lab. Eg Fluor dye. Denat and hyb= lab in cult cell! Binds at posit of gene on chrom. On both chromatids if replicated. Can do dis specif probes with contr present on both chromatids. Dis probe miss from 1=delet. Can probe more than 1 gene. Can targ telom, centromere etc.
- chrom painting- lab all chroms diff col. Range specif DNA sections of chrom 1 all lab with specif probes- fan proks. Show locat chrom 1 in cell. Can show multip transloc eg in tum.
- why use FISH- investig gene in situ eg delet/duplic/transloc. Eg fusion gene. Investig chrom struc eg delet/duplic/transloc. Investig chrom numb eg chrom paint. Investig chrom behav eg anaphase lag- one chrom stays behind.
- genet testing ethic consid- huntingtons eg. 27 yo preg. Fam hx HD on mum side. My 49yo. Req HD test on unborn. Autos domin so if pos then mum has it= decr life expectancy. Who wants to know?
- gene editing CA tx.
LO6: use approp techs to anal and diag dis states.
LO2: imp of PCR.
-detec of mut- most hum mut are 1bp substitut that =sing AA substitut. Prob to detec 1 BP change in 3000m. Hum genome has been cloned and seq. For lot genes of clinic int, precise alt DNA char for comm muts. Eg SC A to T. Test for using allele specif probes in hyb exps. Most DAN diag relies on PCR for amplif of DNA segm arnd mut. Detec mut could foll form eg:
Info on loss/gain RS in PCR prod (eg SC no MstII site). Info on size PCR prod eg detec 3bp delet in CF. presence/absence PCR prod eg allele specif PCR. DNA seq of PCR prod. Hyb with allele specif oligont probes.
-for in dis causing mut allele specif PCR. 1 comm primer with 2 diff allele specif in which 3’ base corresp to base fnd in only 1 allele. PCR prod only amplif if allele specif primer perfectly att with templ DNA at 3’.
-not all mut eas detec by PCR- eg partial gene inversion. Eg some haemophilia A. Need investig gross organisation of genes. S blott allows investig indiv gene in backg of all others. Good for anal larger segms of DNA in and arnd gene. Also used to anal triplet rep disorders eg HD, fragile X synd.
-array comp genom hyb- screen for sub microsc chrom delet if locat not delineated from phenotype. Array of DNA probes cov ent genomes into sol of ma. Pt and norm DNA each lab diff col Fluor tag. Eq amnt hyb to probe array. Hyb sigs detec and comp. for probes where sig of contr exceeds pt= delet.
ANALYSE GENE LEV- RA, cloning, glee elc, PCR last lecture.
ANALYSE DNA MOLEC LEV:
LO1: desc molec basis of DNA seq.
- Sanger chain termin 1975. DdNTPs, each diff col lab. Now automated= genomics- study ent genomes. 3000mn nt hum seq pub 03. Comp gorilla. Also hum microbiome- from diff tiss, comp healthy vs unhealthy.
- NGS- welcome trust Sanger instit 600Gb raw seq per wk per machine= 200 hum genomes. 1000 genomes proj maps 95% all gene vars. 100000 genomes proj CA/rare dis. Cost decr.
- Ethic consid- who int in genome info- dam, partner, dr, gov, police, schools, insurance. Can knowing help people prevent illness later, and how? Will it=discrimin. Who owns seq. Direct to consumer genet test eg paternity, sportsgene, predisp etc.
