بتعرف ال PCR

موضوع: بتعرف ال PCR الثلاثاء سبتمبر 09, 2008 11:29 pm

The polymerase chain reaction

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

PCR principles and procedure
PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents.These components include:

DNA template that contains the DNA region (target) to be amplified.
Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2 is used, but Mn2 can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2 concentration increases the error rate during DNA synthesis
Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 10-200 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

المصدر :http://en.wikipedia.org/wiki/Polymerase_chain_reaction confused

عضو ذهبي المساهمات : 211 تاريخ التسجيل : 28/05/2008

السلام عليكم ورحمة الله وبركاته...
PCR هو technique دقيق جدا وحساس في التعامل معه,والتطبيقات عليه كثيرة...
وإن شاء الله نتحدث عن باقيtechniques المستخدمة في مجال الوراثة......
لكن اسمح لي لقد قمت ببعض التعديلات على الموضوع من حيث الخط والتسنيق ....
ولي طلب أخي الكريم- بارك الله فيك- أن تتحرى اللغة العربية بقدر الإمكان..أعرف أنه من الصعب أحيانا أن نترجم كل الموضوع للغة العربية,
لكن يمكنك أن تلخصه بشكل بسيط ,لأنه هناك ماهو مستوى أول وثاني في التحاليل الطبية..وقد يكونوا أول مرة يسمعوا في الـPCR...

شكرا لك أخ ساري على هذه المشاركة المفيدة...
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