DNA Hyb Flashcards
S blott
-S blott- RE dig. Sep frags gel elec. transf to nylon. Hyb filt with lab gene probe. Detec by expos filt to X Ray.
Anal genom DNA or cloned gene/PCR prod.
-transf to sol supp through nylon/nitrocellulose filter. Gel soaked in alk sol=denat to ssDNA then transf to mem. Capills ct of gel onto filt simplest way. Alternative elecphoretic transf-volt X set up so DNA from gel to filter. Then hyb lab probe. Probe from cloned DNA or oligont synth dNTPs. Then wash filter and detec probe by photog film or Fluor detec.
- why use S blott- investig genes struc eg large delet/duplic. Investig gene expans, triplet reps eg fragile X, HD. Investig mut in genet tests- allele specif probes eg SCD. Investig var, genet relat- DNA fingerprinting.
Allow detec pieces DNA from complex mix. Alos detec v small amnts not visib by stain gel. Comb PCR detec gene struc, expan, reps, mut, var etc.
-blott probes NOT 100% simil to targ seq. 80% still bind, less tight. Still bind if part overlap eg only bind 3’. Probes not aff posit of targ seq on gel.
Sanger dideoxy
- ddNTP NO 3’OH- so DNAP can’t add another dNTP. Used as substr but incorporat blocks elong as prev Pdiest. DNAP new str 5’ to 3’. 5’P on dNTP norm Pdiest to 3’OH in str.
- Denat DNA and add primer= synth. Tube 37 deg cont all dNTPs and 1 ddNTP= stops growth at that base. Length deps on where ddNTP incorp- will be mixt.
- 4 Sep tubes, unique ddNTP and lab primer to init synth. After incub, prods on gel, Sep lanes for each tube. Read seq from bott of gel.
- Fluor ddDNA seq- now we Fluor lab ddNTPs all togeth= diff cols. Diff length frags Sep on thin capill gel and as fall off end data by laser= chromatogram. Direc read seq from peaks. Now get 500-1000bp per seq run.
DNA hyb
-denat by heat/alk. Renat-Complem H bond. Can hyb specif lab probe. Blott comb glee elec and hyb. S blott DNA, N blott RNA. W blott prot.
