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Pre-PCR Processing, PCR Facilitators and Stabilizer Additives

Tekin, Kemal; Kocaman, Mustafa


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{
  "inLanguage": {
    "alternateName": "tur", 
    "@type": "Language", 
    "name": "Turkish"
  }, 
  "description": "<p><strong>&Ouml;zet</strong></p>\n\n<p>Polimeraz Zincir Reaksiyonu (PCR) temelli testler g&uuml;n&uuml;m&uuml;zde enfeksiyon hastal\u0131klar\u0131n\u0131n tan\u0131 ve izleminde en s\u0131k kullan\u0131lan molek&uuml;ler y&ouml;ntemlerdir. PCR&rsquo;\u0131n farkl\u0131 ama&ccedil;lar i&ccedil;in tasarlanm\u0131\u015f nested PCR, ters transkripsiyon PCR, multipleks PCR, real-time PCR ve kantitatif PCR gibi &ccedil;ok say\u0131da farkl\u0131 modifikasyonu geli\u015ftirilmi\u015ftir. Real-time PCR&rsquo;\u0131n bile birbirinden farkl\u0131 g&ouml;r&uuml;nt&uuml;leme y&ouml;ntemleri ve prob tasar\u0131mlar\u0131n\u0131n kullan\u0131ld\u0131\u011f\u0131 &ccedil;ok say\u0131da alt tipi vard\u0131r. PCR y&ouml;ntemi bu modifikasyonlar\u0131 d\u0131\u015f\u0131nda yeni nesil dizi analizi, DNA &ccedil;ip teknolojisi, ters hibridizasyon teknikleri, klonlama &ccedil;al\u0131\u015fmalar\u0131, lumineks teknolojisi, PCR-ELISA ve PCR-RFLP gibi temel molek&uuml;ler tekniklerin bir par&ccedil;as\u0131 veya &ouml;n basama\u011f\u0131 olarak da yayg\u0131n kullan\u0131m alan\u0131 bulmu\u015ftur. Bu &ouml;nemli teknik &ccedil;evresel, klinik, fosil, adli t\u0131p, g\u0131da, bitki, su ve in-vitro ortam &ouml;rneklerinde ba\u015fl\u0131ca DNA veya RNA varl\u0131\u011f\u0131n\u0131 saptamay\u0131 ve analiz etmeyi hedefler. Bu &ouml;rneklerin baz\u0131lar\u0131 PCR &uuml;zerine de\u011fi\u015fen derecelerde inhibit&ouml;r etkiler g&ouml;steren &ccedil;ok say\u0131da kompleks biyolojik molek&uuml;l i&ccedil;eren kar\u0131\u015f\u0131mlard\u0131r. Bu heterojen kar\u0131\u015f\u0131mlar i&ccedil;erisinde bulunan inhibit&ouml;r maddelerin ve ayr\u0131ca DNA ve RNA yap\u0131s\u0131n\u0131 bozabilen enzimlerin uzakla\u015ft\u0131r\u0131lmas\u0131 &ouml;nemlidir. N&uuml;kleik asit eldesi i&ccedil;in baz\u0131 durumlarda h&uuml;cre duvar\u0131 yap\u0131lar\u0131n\u0131 par&ccedil;alamak veya bu molek&uuml;llere ba\u011fl\u0131 proteinleri uzakla\u015ft\u0131rmak gerekirken, t&uuml;m i\u015flemleri DNA ve RNA b&uuml;t&uuml;nl&uuml;\u011f&uuml;n&uuml; bozmadan yapmak &ouml;nemlidir. Yetersiz n&uuml;kleik asit ekstraksiyonu veya inhibit&ouml;r maddelerin varl\u0131\u011f\u0131 &ouml;zellikle az say\u0131da hedef n&uuml;kleik asit i&ccedil;eren &ouml;rneklerde yanl\u0131\u015f negatif sonu&ccedil;lara neden olabilen &ouml;nemli bir problemdir. G&uuml;n&uuml;m&uuml;zde DNA ve RNA eldesi i&ccedil;in &ccedil;ok say\u0131da farkl\u0131 ekstraksiyon ve safla\u015ft\u0131rma y&ouml;ntemi tan\u0131mlanm\u0131\u015f ve bu y&ouml;ntemlerin farkl\u0131 kombinasyonlar\u0131 otomatize sistemlere entegre edilmi\u015ftir. PCR reaksiyonunun verimlili\u011fini art\u0131rman\u0131n alternatif bir yolu ise reaksiyon kar\u0131\u015f\u0131m\u0131na amplifikasyon kolayla\u015ft\u0131r\u0131c\u0131lar\u0131 veya PCR g&uuml;&ccedil;lendiricileri olarak adland\u0131r\u0131lan stabilizat&ouml;r katk\u0131lar\u0131n eklenmesidir. PCR temelli yeni bir protokol tasarlan\u0131rken veya yeni geli\u015ftirilen PCR temelli bir teknikte farkl\u0131 molek&uuml;ller kullan\u0131l\u0131rken ortaya &ccedil;\u0131kabilecek bilinmeyen yeni inhibit&ouml;r etkileri izleyerek PCR &ouml;ncesi ko\u015fullar\u0131n ve amplifikasyon s&uuml;recinin optimize edilmesi &ouml;nem arz etmektedir. Bu makalede her biri farkl\u0131 avantaj ve dezavantajlara sahip PCR &ouml;ncesi haz\u0131rl\u0131k i\u015flemlerinin ve yayg\u0131n kullan\u0131lan amplifikasyon kolayla\u015ft\u0131r\u0131c\u0131lar\u0131n\u0131n genel &ouml;zelliklerinin bir &ouml;zeti sunulmu\u015ftur.</p>\n\n<p><strong>Abstract</strong></p>\n\n<p>Polymerase Chain Reaction (PCR) based tests are the most commonly used molecular methods in the diagnosis and monitoring of infectious diseases today.&nbsp;Numerous different modifications of PCR have been developed and designed for different purposes, such as nested PCR, reverse transcription PCR, multiplex PCR, real-time PCR and quantitative PCR.&nbsp;Even real-time PCR has many types using various imaging methods and different probe designs.&nbsp;Apart from these modifications, the PCR method is also widely used as a part or a preliminary step of basic molecular techniques such as next-generation sequence analysis, DNA chip technology, reverse hybridization techniques, cloning studies, luminex technology, PCR-ELISA and PCR-RFLP.&nbsp;This important technique aims to detect and analyze the presence of DNA or RNA in environmental, clinical, fossil, forensic, food, plant, water and in-vitro media samples.&nbsp;Some of these samples are mixtures containing many complex biological molecules with varying degrees of inhibitory effects on PCR. It is important to remove inhibitory substances in these heterogeneous mixtures as well as enzymes that can disrupt DNA and RNA structure.&nbsp;In order to obtain nucleic acids, in some cases, it is necessary to break down cell wall structures or remove the proteins tightly bound to these molecules and it is important to carry out all these processes without disrupting the integrity of DNA and RNA.&nbsp;Insufficient nucleic acid extraction or the presence of inhibitory agents are important problems that can cause false negative results, especially in samples containing a small number of target nucleic acids.&nbsp;Today, many different extraction and purification methods have been defined for DNA and RNA extraction and different combinations of these methods have been integrated into automated systems.&nbsp;An alternative way to increase the efficiency of the PCR reaction is to add stabilizer additives called amplification facilitators or PCR enhancers to the reaction mixture. It is important to optimize the pre-PCR conditions and amplification process by observing unknown inhibitory effects that may occur when designing a new PCR protocol or using different molecules in a newly developed PCR-based technique. This article provides a summary of the pre-PCR preparation processes, each with different advantages and disadvantages, and the general features of commonly used amplification facilitators.</p>", 
  "license": "https://creativecommons.org/licenses/by/4.0/legalcode", 
  "creator": [
    {
      "affiliation": "Department of Medical Microbiology, UHS Gulhane Training and Research Hospital, Ankara, Turkey", 
      "@id": "https://orcid.org/0000-0002-6610-6540", 
      "@type": "Person", 
      "name": "Tekin, Kemal"
    }, 
    {
      "affiliation": "Tissue Typing Laboratory, UHS Gulhane Training and Research Hospital, Ankara, Turkey", 
      "@id": "https://orcid.org/0000-0002-8217-3970", 
      "@type": "Person", 
      "name": "Kocaman, Mustafa"
    }
  ], 
  "headline": "Pre-PCR Processing, PCR Facilitators and Stabilizer Additives", 
  "image": "https://zenodo.org/static/img/logos/zenodo-gradient-round.svg", 
  "datePublished": "2020-07-22", 
  "url": "https://zenodo.org/record/4548268", 
  "keywords": [
    "Kolayla\u015ft\u0131r\u0131c\u0131, \u0130nhibit\u00f6r, Amplifikasyon, Optimizasyon", 
    "Facilitator, Inhibitor, Amplification, Optimization"
  ], 
  "@context": "https://schema.org/", 
  "identifier": "https://doi.org/10.46683/jmvi.2020.8", 
  "@id": "https://doi.org/10.46683/jmvi.2020.8", 
  "@type": "ScholarlyArticle", 
  "name": "Pre-PCR Processing, PCR Facilitators and Stabilizer Additives"
}
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