{"id":1466,"date":"2020-11-12T18:26:40","date_gmt":"2020-11-12T17:26:40","guid":{"rendered":"http:\/\/atlas-parazitov.uvlf.sk\/?p=1466"},"modified":"2020-11-29T16:33:09","modified_gmt":"2020-11-29T15:33:09","slug":"molekularne-metody","status":"publish","type":"post","link":"https:\/\/atlas-parazitov.uvlf.sk\/?p=1466","title":{"rendered":"Molekul\u00e1rne met\u00f3dy"},"content":{"rendered":"<p>Na presn\u00fa druhov\u00fa identifik\u00e1ciu parazitov sa pou\u017e\u00edva PCR met\u00f3da (<em>polymerase chain reaction<\/em>). Patr\u00ed k&nbsp;naj\u010dastej\u0161ie pou\u017e\u00edvan\u00fdm molekul\u00e1rno-biologick\u00fdm met\u00f3dam. Je to jednoduch\u00e1 met\u00f3da, ktorej princ\u00edpom je namno\u017eenie (amplifik\u00e1cia) ur\u010dit\u00e9ho \u00faseku g\u00e9nu zo vzorky DNA.<\/p>\n<p>Na vykonanie PCR potrebujeme: vzorku vyizolovanej DNA, primery (s\u00fa to kr\u00e1tke \u00faseky synteticky pripraven\u00fdch oligonukleotidov o d\u013a\u017eke 20-30 nukleotidov, ktor\u00e9 nasadaj\u00fa na za\u010diatku a&nbsp;na konci detekovan\u00e9ho \u00faseku DNA), termostabiln\u00fa DNA-polymer\u00e1zu,&nbsp;tlmiv\u00fd roztok s&nbsp;Mg2+ a&nbsp;prekurzory DNA v&nbsp;rovnakom pomere dNTP (dATP, dCTP, dTTP a&nbsp;dGTP). V\u0161etky komponenty do PCR reakcie sa&nbsp;pripravuj\u00fa v&nbsp;mikrosk\u00famavke, ktor\u00fa potom vlo\u017e\u00edme do pr\u00edstroja \u2013 term\u00e1lneho cykl\u00e9ru. Amplifik\u00e1cia DNA prebieha v&nbsp;troch na seba nadv\u00e4zuj\u00facich krokoch, ktor\u00e9 sa cyklicky opakuj\u00fa.<\/p>\n<ol>\n<li>a) Denatur\u00e1cia \u2013 tepeln\u00e1 denatur\u00e1cia (oddelenie dvoch vl\u00e1kien od seba) pri teplote 95\u00b0C<\/li>\n<li>b) Hybridiz\u00e1cia (Anel\u00e1cia) \u2013 hybridiz\u00e1cia (nasadanie) primerov s templ\u00e1tovou DNA na komplement\u00e1rnych miestach pri teplote cca 45-65 \u00b0C<\/li>\n<li>c) Polymeriz\u00e1cia \u2013 zv\u00fd\u0161en\u00edm teploty na 72\u00b0C pomocou Taq-polymer\u00e1zy sa syntetizuje komplement\u00e1rne vl\u00e1kno z&nbsp;vo\u013en\u00fdch dNTP<\/li>\n<\/ol>\n<p>Detekciu z\u00edskan\u00e9ho PCR produktu vykon\u00e1vame pomocou elektrofor\u00e9zy na agar\u00f3zovom g\u00e9ly a&nbsp;vizualizujeme farben\u00edm a&nbsp;o\u017eiaren\u00edm UV svetlom.<\/p>\n<p>Na identifik\u00e1ciu druhov sa pou\u017e\u00edvaj\u00fa aj in\u00e9 vari\u00e1cie PCR met\u00f3dy: ako napr. Nested-PCR, multiplex PCR, PCR\/RFLP, real-time PCR alebo RT-PCR.<\/p>\n<p>V\u00fdsledn\u00fd produkt PCR m\u00f4\u017eeme sekvenova\u0165 Sangerovou met\u00f3dou pomocou sekven\u00e1tora a&nbsp;z\u00edskame sekvencie \u00fasekov konkr\u00e9tnych g\u00e9nov, ktor\u00e9 porovn\u00e1vame so sekvenciami g\u00e9nov v&nbsp;datab\u00e1ze NCBI (GenBank), pri\u010dom&nbsp;z\u00edskame inform\u00e1ciu o&nbsp;ak\u00fd druh parazita sa jedn\u00e1.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Na presn\u00fa druhov\u00fa identifik\u00e1ciu parazitov sa pou\u017e\u00edva PCR met\u00f3da (polymerase chain reaction). Patr\u00ed k&nbsp;naj\u010dastej\u0161ie pou\u017e\u00edvan\u00fdm molekul\u00e1rno-biologick\u00fdm met\u00f3dam. Je to jednoduch\u00e1 met\u00f3da, ktorej princ\u00edpom je namno\u017eenie (amplifik\u00e1cia) ur\u010dit\u00e9ho \u00faseku g\u00e9nu zo vzorky DNA. Na vykonanie PCR potrebujeme: vzorku vyizolovanej DNA, primery (s\u00fa to kr\u00e1tke \u00faseky synteticky pripraven\u00fdch oligonukleotidov o d\u013a\u017eke 20-30 nukleotidov, ktor\u00e9 nasadaj\u00fa na za\u010diatku&#8230;<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"ngg_post_thumbnail":0},"categories":[1],"tags":[],"_links":{"self":[{"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/posts\/1466"}],"collection":[{"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=1466"}],"version-history":[{"count":3,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/posts\/1466\/revisions"}],"predecessor-version":[{"id":1553,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=\/wp\/v2\/posts\/1466\/revisions\/1553"}],"wp:attachment":[{"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1466"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1466"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/atlas-parazitov.uvlf.sk\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1466"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}