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{ | ||
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"cssTheme": "Minimal", | ||
"enabledCssSnippets": [ | ||
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--- | ||
title: "Metódy molekulárnej biológie" | ||
--- | ||
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## Elektroforézy | ||
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### Agarózová gélová EF | ||
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Separácia `DNA` a `RNA`, migrujú v *elektrickom poli* cez *agarózový gel* ku **anóde** (lebo sú záporné) | ||
Rozdeľujú sa podľa *veľkosti molekuly* (ako veľmi sa zasekávajú, pomer m/z je cca rovnaký) | ||
- menšie rýchlejšie | ||
- väčšie pomalšie | ||
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Vizualizácia => **interkalačné činidlá** - viažu sa do veľkého žliabku DNA (mutagény) - `etídium bromid` | ||
Potom *UV vizualizácia* -> naviazané farbivá svietia | ||
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Na to aby sme vedeli cca veľkosť sa používa tzv. `leader` -> známe veľkosti (2, 4, 6, 8, 10kb) | ||
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Najredší gél je 0.7% | ||
Pri menších veľkostiach sa musí používať hustejší gél a niekedy aj kapiláry. | ||
Napr. pri sekvenovaní sa používajú 300b dlhé fragmenty | ||
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Používajú sa aj farbivá na estimation toho nakoľko už proces prešiel, putujú cca ako DNA | ||
-> brómfenolová modrá, xyléncyanol | ||
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Pri **plazmidoch** sa tvoria *3 bandy* -> (kruhová, lineárna a superšpiralizovaná) | ||
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Pri RNA treba denaturovať *sekundárne štruktúry* -> formamid, močovina | ||
Pri separácii RNA z bunky sa objavujú 2 veľké bandy -> *rRNA* | ||
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Extrakcia z gélu sa robí buď vyrezaním alebo rozpustením | ||
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> PFGE?? pulse field electrophoresis | ||
### EMSA | ||
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-> Electrophoretic mobility shift assay | ||
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Väzba proteíny na NK -> spomalenie migrácie | ||
NK značené radioaktívne cez (fosfor P-32) | ||
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### PAGE | ||
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-> Polyakrylamidová gélová elektroforéza | ||
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Separácia proteínov | ||
Pridáva sa SDS (sodium dodecylsulphate) na denaturáciiu a udelenie náboja proteínom | ||
Gél => zmes akrylamidu a bisakrylamidu | ||
Je to vertikálny gél | ||
Farbí sa s Coomassie R-250 Brilliant Blue | ||
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!!! - mutacie sa oznacuju (originalna baza) + miesto + (nova baza) | ||
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#### 2D Page | ||
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1. rozmer: *nedenaturačná* izoelektrická fokusácia (podľa izoelektrického bodu) | ||
2. rozmer: *denaturačné* page | ||
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Izoelektrická fokusácia funguje cez | ||
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![](attachments/izoelektrická-fokusácia.png) | ||
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## Bloty | ||
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Prenos separovaných NK alebo prot na membránu -> špecifická próba alebo protilátka -> detekcia | ||
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- Southern blot: DNA | ||
- Northern blot: RNA | ||
- Western blot: proteíny | ||
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### Northern blot | ||
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Prenos RNA z gélu na *kladne nabitú nylonovú membránu* (elektricky alebo kapilárne) | ||
Detekcia sa robí *hybridizáciou* (RNA komplementarita) s radioaktívne alebo inak značenou próbou | ||
Vizualizácia expozíciou na film alebo fosforovým skrínom | ||
Špecificita záleží na *teplote a salinite* (vyššia pri -> ⬆️ teplota, ⬇️ salinita) | ||
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### Southern blot | ||
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Používa sa väčšinou na overenie vloženia sekvencie do génomu | ||
Najprv sa DNA reštrikčne naštiepi | ||
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### Western blot | ||
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Presun z PAGE gélu na nitrocelulózovú alebo PVDF membránu | ||
Na rozoznávanie sa používajú *protilátky* - monoklonálne (špecifické) vs polyklonálne | ||
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Vizualizácia (podľa toho čo je naviazané na sekund. protil.): | ||
- kolorimetrická - enzým, ktorý štiepi farbivo | ||
- chemiluminiscencia - enzým, ktorý chemicky tvorí svetlo | ||
- fluorescencia - fluorescenčné farbivo | ||
> [Stokesov posuv](https://en.wikipedia.org/wiki/Stokes_shift) | ||
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## Reštrikčné endonukleázy | ||
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Enzýmy ktoré štiepia DNA na určitých miestach podľa sekvencie | ||
Štiepia *palindromické sekvencie* a stále symetricky (lebo sú to vlastne dimérne enzýmy) | ||
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Môžu tvoriť: | ||
- blunt konce | ||
- sticky konce | ||
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Fok1 - štiepi 9bp a 13bp downstream od rozoznávaného miesta |
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## Stavba | ||
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### Bunková stena prok. | ||
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Mechanická odolnosť, udržujú turgor | ||
Tvorená peptidoglykánom, sieťová štruktúra | ||
Cieľom mnohých antibiotík (betalaktamove, cefalosporiny) a lyzozym -> štiepia BS -> pukne | ||
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#### Gramove farbenie | ||
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Delenie: *Gram +/-* | ||
Na základe farbenia kryštal. violeťou + safranin a fuchsin | ||
Gram + -> veľa peptidoglykanu -> zachytava violeť -> *fialove* | ||
Gram - -> málo peptidoglykanu -> nezachytava violet -> *cervena* (safranin a fuchsin farbia vnutro) | ||
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Gram negativne maju aj lipopolysacharidovú vrstvu | ||
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#### Glykokalyx | ||
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Polysacharidovy povrch u niektorych bakterii | ||
Biofilm, ochrana pred fagocytozou makrofagov | ||
Cielom protilatok | ||
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### Výbežky | ||
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#### Bičík | ||
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#### Fimbrie | ||
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Prichytenie bakterii k povrchu -> biofilm | ||
Interakcie na povrchu s cielovymi bunkami -> rozne epitely | ||
Mechanizmus virulencie | ||
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#### Pili | ||
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Zabezpecenie konjugacie medzi dvoma bakteriami -> HGT plazmidu | ||
Oligomérny proteín pilín | ||
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### Cytoplazma | ||
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#### Ribozómy | ||
30S + 50S => 70S | ||
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#### Plynové vakuoly | ||
Z hydrofobnych proteinov, nepriestupne pre vody | ||
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#### Tylakoidy | ||
Jedine membranove struktury prokaryotov -> typicke pre sinice | ||
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## Tvar | ||
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![|600](attachments/tvary-baktérií.png) | ||
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## Rozmnožovanie | ||
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#### Endospóra | ||
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![|800](attachments/endospora.png) |
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