واکنش زنجیره‌ای پلیمراز: انواع، کاربرد‌ها، ابزارهای بیوانفورماتیک و راهنمای طراحی آغازگر با تأکید بر غلات

نوع مقاله : مروری

نویسندگان

1 گروه تولید و ژنتیک گیاهی دانشکده کشاورزی دانشگاه ارومیه. ارومیه، ایران.

2 گروه بیوتکنولوژی کشاورزی دانشکده کشاورزی دانشگاه تربیت مدرس. تهران، ایران.

چکیده

مقدمه: واکنش زنجیره­ای پلیمراز (PCR) یک فناوری بسیار مهم و کاربردی در زمینه­های مختلف علوم زیستی و پزشکی می‌باشد. با استفاده از این تکنیک می‌توان در شرایط درون‌شیشه‌ای (in vitro) با حداقل مقدار DNA، میلیون­ها نسخه از یک الگوی مشخص را تکثیر نمود. این امر پیشرفت­های عمده‌ای در زیست‌‌‌‌شناسی مولکولی و پزشکی رقم زده است. این تکنیک، کاربرد‌های بسیار متنوعی در علوم زیستی دارد. آغازگرهای PCR جزء جدایی‎ناپذیر PCR هستند. آغازگرهایی که به خوبی طراحی شوند، به سیستم PCR اجازه می­دهند تا با دقت کار کند. برای ایجاد یک جفت آغازگر خوب PCR، عوامل متعددی از جمله طول جفت باز، دمای ذوب و اتصال، تکرار جفت باز و غیره باید در نظر گرفته شوند. آغازگرهای PCR که با دقت طراحی می‌شوند، نه­ تنها حساسیت و اختصاصیت را افزایش می­دهند، بلکه تلاش صرف شده برای بهینه­سازی تجربی را نیز کاهش می­دهند؛ در حالیکه PCR به یکی از با ارزش ترین تکنیک هایی تبدیل شده است که در حال حاضر در علوم زیستی، تشخیصی و پزشکی قانونی استفاده می شود.
PCR has become one of the most valuable techniques currently used in life sciences, diagnostics and forensics.
PCR به یکی از با ارزش ترین تکنیک ها تبدیل شده است که در حال حاضر در علوم زیستی، تشخیصی و پزشکی قانونی مورد استفاده قرار می گیرد.
PCR has become one of the most valuable techniques currently used in life sciences, diagnostics and forensics.
«نتایج کامل» بار نشد
امتحان مجدد
درحال تلاش مجدد…
طراحی ضعیف آغازگر‌ همراه با عدم بهینه­سازی شرایط واکنش احتمالاً منجر به کاهش دقت فنی و تشخیص مثبت یا منفی کاذب در تکثیر قطعات DNA هدف می­شود. بیوانفورماتیک نه تنها برای تحقیقات پایه، بلکه برای تحقیقات کاربردی در علوم زیستی به ابزاری اساسی تبدیل شده است. ابزارهای طراحی آغازگر‌ با خودکارسازی محاسبات پیچیده و دقیق در کمترین زمان ممکن طراحی بهینه و کارآمد آغازگر‌ها را فراهم می­کنند.
مواد و روش‌ها: مقاله حاضر یک مقاله مروری می­باشد که به شیوه تحلیل محتوا (Content analysis) با جستجوی کلید واژه­های واکنش زنجیره­ای پلی­مراز (PCR)، انواع PCR، طراحی آغازگر برای PCR در مقاله­های مرتبط در پایگاه­های اینترنتیGoogle scholar ،Web of science ،PubMed  و Scopus به دست آمده است.
یافته‌ها: این مقاله قصد دارد با مروری بر روش PCR، انواع و کاربردهای آن و با استفاده از پیوندها و خدمات موجود در وب، راهنمایی‌ تقریباً کاملی به منظور طراحی آغازگر‌های کارآمد و بهینه برای آنالیز‌های PCR و qPCR ارائه نماید. در این راستا، در قسمت اول مقاله انواع روش­ها از قبیل PCR استاندارد، PCR رونویسی معکوس (RT- PCR)، PCR کمی (qPCR)، RT-PCR/qPCR ترکیبی و ... با ارایه شکل‏های مناسب توضیح داده شده­ است. در ادامه کاربرد انواع PCRها با تأکید بر تحقیقات غلات خلاصه شده است. در انتها، روش­های طراحی آغازگر خصوصاً برای qPCR، طبقه­بندی و بررسی نرم­افزارهای موجود و قابلیت­های آن‎ها، روش­ها و نرم­افزارهای بررسی خصوصیات آغازگرها برای کسب حداکثر اختصاصیت و کارایی PCR، نحوه استخراج توالی ژن در طراحی آغازگر برای PCR با ارایه مثال­های عملی ارایه شده است.
نتیجه‌گیری: یک PCR با آغازگرهای ضعیف به دلیل تکثیر غیر اختصاصی و یا تشکیل دیمر آغازگر، می­تواند محصول کمی داشته باشد یا اصلاً محصولی به دنبال نداشته باشد. ابزار‌های آنلاین متعددی معرفی شدند که برای تهیه آغازگرهای مؤثر برای PCR در زمینه زیست مولکولی استفاده می‌شوند.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Polymerase Chain Reaction: Types, Applications, Bioinformatics Tools and Guideline for Primer Design with emphasis on cereals

نویسندگان [English]

  • Mona Bordbar 1
  • Reza Darvishzadeh 1
  • Morad Jafari 1
  • Danial Kahrizi 2
1 Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran.
2 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modarres University, Tehran, Iran.
چکیده [English]

Introduction: Polymerase chain reaction (PCR) is a critical and practical technology in various biological and medical sciences fields. Using this technique, millions of copies of a specific pattern can be reproduced in vitro with a minimal amount of DNA. This has made significant advances in molecular biology and medicine. This technique has many applications in biological sciences. PCR primers are an integral part of PCR. Well-designed primers allow the PCR system to work accurately. To create a good PCR primer pair, several factors must be considered, including base pair length, melting and annealing temperature, base pair repeats, etc. Carefully designed PCR primers increase sensitivity and specificity and reduce the effort spent on experimental optimization. The poorly designed primer and the lack of optimization of the reaction conditions probably lead to decreased technical accuracy and false positive or negative detection in the amplification of the target DNA fragments. Bioinformatics has become an essential tool for basic research and applied research in life sciences. Primer designing tools provide optimal and efficient design of primers by automating complex and accurate calculations in the shortest possible time.
Materials and methods: This review article is a content analysis study performed by searching Polymerase Chain Reaction (PCR), PCR type, and primer design for PCR in related articles on Google Scholar, Web of Science, PubMed and Scopus.
Results: This article aims to provide an almost complete guide for designing efficient and optimal primers for PCR and qPCR analyses by reviewing the PCR method, its types and applications and using the links and web-based services. In this regard, in the first part of the article, various methods, such as standard PCR, reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), combined RT-PCR/qPCR, etc., will be explained with suitable figures. The following summarizes the use of PCR types, emphasizing cereal research. In the end, primer design methods, especially for qPCR, classification and review of existing software and their capabilities, methods and software for testing the features of primers to obtain maximum specificity and PCR efficiency, extracting the gene sequence for PCR primer designing is presented with practical examples.
Conclusion: A PCR with weak primers can produce little or no product due to non-specific amplification or primer dimer formation. Several online tools were introduced that are used to prepare effective primers for PCR in the field of molecular biology.

کلیدواژه‌ها [English]

  • Bioinformatics
  • Conventional PCR
  • Real-Time PCR
  • Primer design

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