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Pre-PCR Processing, PCR Facilitators and Stabilizer Additives

Tekin, Kemal; Kocaman, Mustafa


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    <subfield code="a">&lt;p&gt;&lt;strong&gt;&amp;Ouml;zet&lt;/strong&gt;&lt;/p&gt;

&lt;p&gt;Polimeraz Zincir Reaksiyonu (PCR) temelli testler g&amp;uuml;n&amp;uuml;m&amp;uuml;zde enfeksiyon hastalıklarının tanı ve izleminde en sık kullanılan molek&amp;uuml;ler y&amp;ouml;ntemlerdir. PCR&amp;rsquo;ın farklı ama&amp;ccedil;lar i&amp;ccedil;in tasarlanmış nested PCR, ters transkripsiyon PCR, multipleks PCR, real-time PCR ve kantitatif PCR gibi &amp;ccedil;ok sayıda farklı modifikasyonu geliştirilmiştir. Real-time PCR&amp;rsquo;ın bile birbirinden farklı g&amp;ouml;r&amp;uuml;nt&amp;uuml;leme y&amp;ouml;ntemleri ve prob tasarımlarının kullanıldığı &amp;ccedil;ok sayıda alt tipi vardır. PCR y&amp;ouml;ntemi bu modifikasyonları dışında yeni nesil dizi analizi, DNA &amp;ccedil;ip teknolojisi, ters hibridizasyon teknikleri, klonlama &amp;ccedil;alışmaları, lumineks teknolojisi, PCR-ELISA ve PCR-RFLP gibi temel molek&amp;uuml;ler tekniklerin bir par&amp;ccedil;ası veya &amp;ouml;n basamağı olarak da yaygın kullanım alanı bulmuştur. Bu &amp;ouml;nemli teknik &amp;ccedil;evresel, klinik, fosil, adli tıp, gıda, bitki, su ve in-vitro ortam &amp;ouml;rneklerinde başlıca DNA veya RNA varlığını saptamayı ve analiz etmeyi hedefler. Bu &amp;ouml;rneklerin bazıları PCR &amp;uuml;zerine değişen derecelerde inhibit&amp;ouml;r etkiler g&amp;ouml;steren &amp;ccedil;ok sayıda kompleks biyolojik molek&amp;uuml;l i&amp;ccedil;eren karışımlardır. Bu heterojen karışımlar i&amp;ccedil;erisinde bulunan inhibit&amp;ouml;r maddelerin ve ayrıca DNA ve RNA yapısını bozabilen enzimlerin uzaklaştırılması &amp;ouml;nemlidir. N&amp;uuml;kleik asit eldesi i&amp;ccedil;in bazı durumlarda h&amp;uuml;cre duvarı yapılarını par&amp;ccedil;alamak veya bu molek&amp;uuml;llere bağlı proteinleri uzaklaştırmak gerekirken, t&amp;uuml;m işlemleri DNA ve RNA b&amp;uuml;t&amp;uuml;nl&amp;uuml;ğ&amp;uuml;n&amp;uuml; bozmadan yapmak &amp;ouml;nemlidir. Yetersiz n&amp;uuml;kleik asit ekstraksiyonu veya inhibit&amp;ouml;r maddelerin varlığı &amp;ouml;zellikle az sayıda hedef n&amp;uuml;kleik asit i&amp;ccedil;eren &amp;ouml;rneklerde yanlış negatif sonu&amp;ccedil;lara neden olabilen &amp;ouml;nemli bir problemdir. G&amp;uuml;n&amp;uuml;m&amp;uuml;zde DNA ve RNA eldesi i&amp;ccedil;in &amp;ccedil;ok sayıda farklı ekstraksiyon ve saflaştırma y&amp;ouml;ntemi tanımlanmış ve bu y&amp;ouml;ntemlerin farklı kombinasyonları otomatize sistemlere entegre edilmiştir. PCR reaksiyonunun verimliliğini artırmanın alternatif bir yolu ise reaksiyon karışımına amplifikasyon kolaylaştırıcıları veya PCR g&amp;uuml;&amp;ccedil;lendiricileri olarak adlandırılan stabilizat&amp;ouml;r katkıların eklenmesidir. PCR temelli yeni bir protokol tasarlanırken veya yeni geliştirilen PCR temelli bir teknikte farklı molek&amp;uuml;ller kullanılırken ortaya &amp;ccedil;ıkabilecek bilinmeyen yeni inhibit&amp;ouml;r etkileri izleyerek PCR &amp;ouml;ncesi koşulların ve amplifikasyon s&amp;uuml;recinin optimize edilmesi &amp;ouml;nem arz etmektedir. Bu makalede her biri farklı avantaj ve dezavantajlara sahip PCR &amp;ouml;ncesi hazırlık işlemlerinin ve yaygın kullanılan amplifikasyon kolaylaştırıcılarının genel &amp;ouml;zelliklerinin bir &amp;ouml;zeti sunulmuştur.&lt;/p&gt;

&lt;p&gt;&lt;strong&gt;Abstract&lt;/strong&gt;&lt;/p&gt;

&lt;p&gt;Polymerase Chain Reaction (PCR) based tests are the most commonly used molecular methods in the diagnosis and monitoring of infectious diseases today.&amp;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.&amp;nbsp;Even real-time PCR has many types using various imaging methods and different probe designs.&amp;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.&amp;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.&amp;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.&amp;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.&amp;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.&amp;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.&amp;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.&lt;/p&gt;</subfield>
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