{"id":1121,"date":"2016-12-05T21:33:35","date_gmt":"2016-12-05T20:33:35","guid":{"rendered":"http:\/\/www.newslab.sk\/2016\/12\/05\/doba-laboratorna-vyvoj-laboratornych-technik-na-extrakciu-nukleovych-kyselin\/"},"modified":"2017-10-03T14:55:42","modified_gmt":"2017-10-03T12:55:42","slug":"the-laboratory-age","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/the-laboratory-age\/","title":{"rendered":"The laboratory age"},"content":{"rendered":"<pre><strong><span style=\"color: #ff0000;\">*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf <\/span><\/strong>\r\n <strong><span style=\"color: #ff0000;\">attachment at the end of the paper. <\/span><\/strong><\/pre>\n<p>&nbsp;<\/p>\n<p>\u017dijeme v\u00a0modernej dobe laborat\u00f3rnej, v\u00a0dobe, ktor\u00e1 n\u00e1m otv\u00e1ra mo\u017enosti vstupova\u0165 st\u00e1le hlb\u0161ie do sveta molek\u00fal, produkova\u0165 v\u00a0kr\u00e1tkom \u010dase a\u00a0na malom priestore obrovsk\u00e9 mno\u017estvo d\u00e1t. \u00a0V\u00a0laborat\u00f3rnej diagnostike v\u00a0posledn\u00fdch desa\u0165ro\u010diach zaznamen\u00e1vame v\u00fdrazn\u00fd posun k\u00a0automatiz\u00e1cii vy\u0161etrovac\u00edch procesov, minimaliz\u00e1cii zariaden\u00ed a\u00a0po\u010d\u00edta\u010dom asistovanej anal\u00fdze. Tento stav je v\u00fdsledkom sna\u017eenia mnoh\u00fdch zn\u00e1mych v\u00fdskumn\u00edkov aj bezmenn\u00fdch \u010dlenov v\u00fdvojov\u00fdch t\u00edmov.<\/p>\n<p>\u0160vaj\u010diarsky lek\u00e1r, Friedrich Miescher , v\u00a0roku 1869 prv\u00fd kr\u00e1t izoloval DNA. N\u00e1dejal sa , \u017ee objav\u00ed fundament\u00e1lne princ\u00edpy \u017eivota a\u00a0chemick\u00fa podstatu buniek. Spo\u010diatku experimentoval s anal\u00fdzou chemick\u00fdch l\u00e1tok (preva\u017ene prote\u00ednov, ako hlavnej zlo\u017eky cytoplazmy) z\u00a0leukocytov z\u00edskan\u00fdch z\u00a0hnisav\u00fdch obv\u00e4zov. Zdokona\u013eovan\u00edm pokusov sa mu podarilo zisti\u0165, \u017ee dok\u00e1\u017ee z roztoku pridan\u00edm kyseliny precipitova\u0165 dovtedy nepoznan\u00fa substanciu, op\u00e4\u0165 rozpustn\u00fa po pridan\u00ed alkalickej l\u00e1tky. Zaznamenal, \u017ee t\u00e1to substancia je bohat\u00e1 na fosfor a\u00a0vzh\u013eadom na jej lokaliz\u00e1ciu v\u00a0bunke ju nazval nukle\u00edn. V\u00a0roku 1889 ju\u00a0 jeho \u017eiak Richard Altman na podklade jej chemick\u00fdch vlastnost\u00ed premenoval na nukleov\u00fa kyselinu (NK).(1,2)<\/p>\n<p>Napriek nerozpoznan\u00e9mu v\u00fdznamu nukleov\u00fdch kysel\u00edn sa v\u00a0nasleduj\u00facich rokoch\u00a0 o\u00a0ne zv\u00fd\u0161il z\u00e1ujem, zdokona\u013eovali sa izola\u010dn\u00e9 a purifika\u010dn\u00e9 techniky, ale a\u017e v\u00a0roku 1935 sa rusk\u00e9mu vedcovi Andrejovi Nikolajevi\u010dovi Belozersk\u00e9mu podarilo izolova\u0165 \u010dist\u00fa DNA, \u010d\u00edm sa definit\u00edvne otvorila br\u00e1na k\u00a0poznaniu jej \u0161trukt\u00fary a\u00a0funkcie v\u00a0organizme. Poznanie chemickej a\u00a0fyzik\u00e1lnej povahy nukleov\u00fdch kysel\u00edn znamenalo \u010fal\u0161\u00ed rozvoj izola\u010dn\u00fdch techn\u00edk.\u00a0\u00a0\u00a0 (3).<\/p>\n<p>M\u00f4\u017eeme kon\u0161tatova\u0165, \u017ee v\u0161etky historick\u00e9 met\u00f3dy pretrv\u00e1vaj\u00fa, aj ke\u010f v\u00a0upravenej podobe do\u00a0s\u00fa\u010dasnosti. V\u00a0roku 1958, v \u010dase experimentov\u00a0 Meselsona a\u00a0Stahla (dok\u00e1zali semikonzervat\u00edvnos\u0165 DNA) sa na extrahovanie\u00a0 DNA vyu\u017e\u00edvala hustotn\u00e1 gradientov\u00e1 centrifug\u00e1cia, ktor\u00e1 je v\u00a0\u0161peci\u00e1lnych pr\u00edpadoch a\u00a0r\u00f4znych \u00faprav\u00e1ch vyu\u017e\u00edvan\u00e1 dodnes \u00a0Na izol\u00e1ciu nukleov\u00fdch kysel\u00edn\u00a0 sa naj\u010dastej\u0161ie zau\u017e\u00edvali dva pr\u00edstupy \u2013 prv\u00fd (aj historicky),\u00a0 zalo\u017een\u00fd na roztokoch a\u00a0druh\u00fd, zalo\u017een\u00fd na kol\u00f3nkach (nosi\u010doch). V\u00fdber vhodn\u00e9ho protokolu podlieha predov\u0161etk\u00fdm potrebe mno\u017estva, kvality a\u00a0druhu fin\u00e1lnej nukleovej kyseliny, ale aj kvantitat\u00edvnym, priestorov\u00fdm, \u010dasov\u00fd a\u00a0finan\u010dn\u00fdm pomerom. ( 4).<\/p>\n<p>Prv\u00fdm krokom \u00faspe\u0161nej izol\u00e1cie NK je l\u00fdza buniek. Jej\u00a0cie\u013eom je uvo\u013eni\u0165 obsah bunky (resp. organel) pomocou jemn\u00fdch detergentov, pr\u00edpadne ultrazvukom. Aby sa zabr\u00e1nilo degrad\u00e1cii cie\u013eov\u00fdch molek\u00fal, rozpad bunky prebieha v\u00a0tlmivom roztoku a\u00a0vhodn\u00fdch teplotn\u00fdch podmienkach. Na oddelenie cie\u013eovej NK z\u00a0roztoku bunkov\u00e9ho obsahu sa vyu\u017e\u00edva do\u010dasn\u00e1 denatur\u00e1cia \u2013 teplotn\u00e1, vyso\u013eovacia met\u00f3da alebo zr\u00e1\u017eanie organick\u00fdmi rozp\u00fa\u0161\u0165adlami. Kontamin\u00e1ciu DNA zvy\u0161kami RNA je mo\u017en\u00e9 jednoducho odstr\u00e1ni\u0165 pridan\u00edm enz\u00fdmu ribonukle\u00e1zy.<\/p>\n<p>Medzi jednoduch\u0161ie a\u00a0historicky p\u00f4vodnej\u0161ie extrak\u010dn\u00e9 pr\u00edstupy patr\u00ed vyso\u013eovacia met\u00f3da, zalo\u017een\u00e1 na princ\u00edpe zmeny rozpustnosti molek\u00fal DNA v\u00a0z\u00e1vislosti na zmene koncentr\u00e1cie i\u00f3nov v\u00a0roztoku (naj\u010dastej\u0161ie s\u00edranu am\u00f3nneho). \u010eal\u0161ou jednoduchou mo\u017enos\u0165ou je zr\u00e1\u017eanie organick\u00fdmi rozp\u00fa\u0161\u0165adlami (etanol, polyethylenglykol), ktor\u00e9 sa uskuto\u010d\u0148uje na z\u00e1klade ich schopnosti zn\u00ed\u017ei\u0165 rozpustnos\u0165 NK (zv\u00fd\u0161en\u00edm intramolekulovej elektrostatickej interakcie).\u00a0 R\u00f4zna rozpustnos\u0165 DNA sa vyu\u017e\u00edva aj pri fenol-chloroformovej extrakcii. Jej podstatou je oddelenie vodnej f\u00e1zy (s\u00a0 DNA) od roztoku obsahuj\u00faceho fenol a\u00a0chloroform pomocou vysokoot\u00e1\u010dkovej centrifug\u00e1cie, s\u00a0n\u00e1slednou etanolovou precipit\u00e1ciou DNA. Aj ke\u010f je t\u00e1to met\u00f3da \u010dasovo n\u00e1ro\u010dn\u00e1 a\u00a0z\u00e1visl\u00e1 na kvalite prevedenia, je st\u00e1le pomerne \u010dasto vyu\u017e\u00edvan\u00e1 pre jej finan\u010dn\u00fa nen\u00e1ro\u010dnos\u0165 a\u00a0vy\u0161\u0161ie v\u00fd\u0165a\u017eky DNA. (4)<\/p>\n<p>Met\u00f3da RESIN (z angl.; \u017eivica) vyu\u017e\u00edva viazanie a\u00a0n\u00e1sledn\u00e9 uvo\u013enenie po\u017eadovanej substancie pomocou v\u00fdmeny i\u00f3nov medzi pevnou f\u00e1zou a\u00a0kvapalinou obklopuj\u00facou pevn\u00fa f\u00e1zu. Je to pomerne star\u00e1 met\u00f3da (Thomas a\u00a0Way, 1906), spo\u010diatku vyu\u017e\u00edvan\u00e1 v\u00a0anorganickej ch\u00e9mii. P\u00f4vodn\u00e9 pr\u00edrodn\u00e9 materi\u00e1ly \u2013 sulf\u00f3novan\u00e9 uhlie a\u00a0zeolit, boli v\u00a0polovici minul\u00e9ho storo\u010dia nahraden\u00e9 organick\u00fdmi i\u00f3nomeni\u010dmi, ktor\u00e9 sa pou\u017e\u00edvaj\u00fa a\u017e do s\u00fa\u010dasnosti (aj v\u00a0podobe komer\u010dne dostupn\u00fdch kitov). Je to pomerne r\u00fdchla met\u00f3da s\u00a0vysok\u00fdm v\u00fd\u0165a\u017ekom a\u00a0\u010distotou DNA. (4,5)<\/p>\n<p>Met\u00f3da SILIKA matrice je tie\u017e pomerne star\u00e1, aj pri nej nahradil pr\u00edrodn\u00fd materi\u00e1l- diatomit (rozsievkov\u00e1 zemina), synteticky pripraven\u00fd silikag\u00e9l. Silika materi\u00e1l (SiO<sub>2<\/sub>)\u00a0 v\u00a0pr\u00edtomnosti chaotropn\u00fdch sol\u00ed (jodod sodn\u00fd, guanidin thiokyan\u00e1t) adsorbuje DNA na svoj povrch, z\u00a0ktor\u00e9ho je po opakovanom pre\u010disten\u00ed uvo\u013enen\u00e1 pomocou elu\u010dn\u00e9ho \u010dinidla. Je to \u010dasovo nen\u00e1ro\u010dn\u00e1 met\u00f3da\u00a0 s\u00a0vysokou \u010distotou DNA a\u00a0je tie\u017e dostupn\u00e1 vo forme kitov.(4)<\/p>\n<p>Najmlad\u0161ie met\u00f3dy\u00a0 izol\u00e1cie vyu\u017e\u00edvaj\u00fa magnetick\u00e9 mikro\u010dastice alebo \u00a0nano\u010dastice. Maj\u00fa jadro, \u00a0tvoren\u00e9 magnetick\u00fdmi oxidmi \u017eeleza, obalen\u00e9 l\u00e1tkou (organick\u00e9 polym\u00e9ry, silika) umo\u017e\u0148uj\u00facou naviazanie cie\u013eov\u00fdch objektov, napr\u00edklad NK . Princ\u00edpom je separ\u00e1cia tak\u00fdchto magnetick\u00fdch partik\u00fal p\u00f4soben\u00edm siln\u00e9ho magnetu, ich n\u00e1sledn\u00e9 prem\u00fdvanie a\u00a0uvo\u013enenie NK z\u00a0komplexu. \u00a0Aj v\u00a0tomto pr\u00edpade je dostupn\u00fdch nieko\u013eko kitov, ktor\u00e9 umo\u017e\u0148uj\u00fa v\u00a0pomerne kr\u00e1tkom \u010dase z\u00edska\u0165 DNA nekontaminovan\u00fa prote\u00ednmi alebo RNA. Ke\u010f\u017ee t\u00e1to met\u00f3da nevy\u017eaduje manipul\u00e1ciu so \u0161kodliv\u00fdmi organick\u00fdmi rozp\u00fa\u0161\u0165adlami, opakovan\u00fa centrifug\u00e1ciu, filtr\u00e1ciu vo v\u00e1kuu alebo kol\u00f3nkov\u00fa separ\u00e1ciu , je ide\u00e1lna na automatizovan\u00fa separ\u00e1ciu. \u00a0Izola\u010dn\u00e9 automaty zabezpe\u010dia v\u00a0kr\u00e1tkom \u010dase a\u00a0\u0161tandardn\u00fdch podmienkach spravidla v\u00e4\u010d\u0161ie mno\u017estvo vzoriek, preto s\u00fa ide\u00e1lne v\u00a0rutinn\u00fdch aj v\u00fdskumn\u00fdch laborat\u00f3ri\u00e1ch. Ich nev\u00fdhodou je finan\u010dn\u00e1 n\u00e1ro\u010dnos\u0165 (in\u0161trument, \u0161peci\u00e1lne plasty, kity). (4,6)<\/p>\n<p>Automatiz\u00e1cia laborat\u00f3ri\u00ed je nepochybne pr\u00ednosom; redukuje pracovn\u00fd \u010das i\u00a0person\u00e1lne zdroje , zvy\u0161uje kvalitu a\u00a0reproducibilitu v\u00fdsledkov. V\u00a0dne\u0161nej dobe je nevyhnutn\u00e9 pokra\u010dova\u0165 v\u00a0tomto trende. Nesmieme v\u0161ak pod\u013eahn\u00fa\u0165 pohodln\u00e9mu tlaku automatov. Na\u0161e mysle nech s\u00fa rovnako otvoren\u00e9, ako mysle b\u00e1date\u013eov,\u00a0 ktor\u00ed\u00a0 pre n\u00e1s dobu laborat\u00f3rnu otvorili.<\/p>\n<p><u>\u00a0<\/u><\/p>\n<ol>\n<li>Maderspacher F. Rags before the riches: Friedrich Miescher and the discovery of DNA. <em> Biol. <\/em>2004;14(15):R608<\/li>\n<li>Wolf Friedrich Miescher, the man who discovered DNA. <em>Chemical Heritage<\/em>. 2003;21(10-11): 37-41<\/li>\n<li>1900 &#8211; 1953 &#8211; Converging on DNA. <a href=\"http:\/\/www.accessexcellence.org\/RC\/AB\/BC\/1900-1953.php\">http:\/\/www.accessexcellence.org\/RC\/AB\/BC\/1900-1953.php<\/a><\/li>\n<li>Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. <em> Biomed. Biotechnol<\/em>. 2009:1-10<\/li>\n<li>McGarvey FX. <em>Introduction to industrial ion Exchange<\/em>. Sybron Chemicals Inc., Birmingham, New Jersey; 1983<\/li>\n<li>H\u00faska D, Baloun J, Trnkov\u00e1 L, et al. Vyu\u017eit\u00ed paramagnetick\u00fdch \u010d\u00e1stic pro izolaci mRNA. <em>CHEMagaz\u00edn<\/em>. 2008;18(3):14-15<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper. &nbsp; \u017dijeme v\u00a0modernej dobe laborat\u00f3rnej, v\u00a0dobe, ktor\u00e1 n\u00e1m otv\u00e1ra mo\u017enosti vstupova\u0165 st\u00e1le hlb\u0161ie do sveta molek\u00fal, produkova\u0165 v\u00a0kr\u00e1tkom \u010dase a\u00a0na malom priestore obrovsk\u00e9 mno\u017estvo d\u00e1t. \u00a0V\u00a0laborat\u00f3rnej diagnostike v\u00a0posledn\u00fdch desa\u0165ro\u010diach zaznamen\u00e1vame<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[290],"tags":[727,600,726],"class_list":["post-1121","post","type-post","status-publish","format-standard","hentry","category-genetics","tag-dna-history","tag-dna-isolation","tag-nucleic-acids","typ_clanku-review-article"],"acf":{"abstrakt":"<p>Successful isolation of nucleic acids and proteins is a basis of a numerous laboratory, genetical, biochemical, immunological or microbiological tests and examination. At the moment, there is a wide range of extraction methods available and selecting the right one depend on the type and subsequent application of the target molecule. The article offers a historical overview of the development of the respective methods and their application at present.<\/p>\n<p><strong>Kay words:<\/strong> nucleic acids, DNA isolation, DNA history<\/p>\n","casopis":[{"ID":1000,"post_author":"7","post_date":"2015-11-21 14:33:24","post_date_gmt":"2015-11-21 13:33:24","post_content":"<h4><strong>GENETICS<\/strong><\/h4>\r\n<ul>\r\n \t<li>The laboratory age<\/li>\r\n \t<li>The Odyssey of DNA reading<\/li>\r\n \t<li>Comparative genomic hybridisation: a methodological introduction<\/li>\r\n \t<li>Next generation sequencing and its application in clinical genetics<\/li>\r\n<\/ul>\r\n<h4><\/h4>\r\n<h4><strong>BIOCHEMISTRY<\/strong><\/h4>\r\n<ul>\r\n \t<li>Determining of the trace elements in blood serum<\/li>\r\n \t<li>Determining of \u03b1-tocopherol (vitamin E) in serum by way of liquid chromatography with tandem mass spectrometry (LC\/MS\/MS)<\/li>\r\n \t<li>Analysis of urinary calculi and its path to Europe<\/li>\r\n<\/ul>\r\n&nbsp;\r\n<h4><strong>IMMUNOLOGY <\/strong><\/h4>\r\n<ul>\r\n \t<li>New autoantibodies in diagnosis of autoimmunity myopathies<\/li>\r\n \t<li>Taking advantage of flow-based cytometry in determining prognostic markers<\/li>\r\n<\/ul>\r\nCD38 and ZAP-70 in patients with B-CLL\r\n\r\n&nbsp;\r\n<h4><strong>CYTOLOGY AND PATHOLOGY <\/strong><\/h4>\r\n<ul>\r\n \t<li>Mucinous ovarian carcinoma \u2013 news in diagnosis from the pathologist\u2019s perspective<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-12015","to_ping":"","pinged":"","post_modified":"2017-08-16 21:43:10","post_modified_gmt":"2017-08-16 19:43:10","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/casopis\/newslab-12015\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"5","upload_clanok":{"ID":1122,"id":1122,"title":"medirex_1_2015-pavlikova","filename":"MEDIREX_1_2015-\u2013-Pavlikova.pdf","filesize":139310,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2016\/12\/MEDIREX_1_2015-\u2013-Pavlikova.pdf","link":"https:\/\/www.newslab.sk\/en\/the-laboratory-age\/medirex_1_2015-pavlikova\/","alt":"","author":"7","description":"","caption":"","name":"medirex_1_2015-pavlikova","status":"inherit","uploaded_to":1121,"date":"2016-12-05 20:31:33","modified":"2016-12-05 20:31:33","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1121","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1121"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1121\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1121"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1121"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1121"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}