PCR is shorthand for a simple but very useful procedure in molecular biology called the polymerase chain reaction. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies. In other words, PCR enables you to produce millions of copies of a specific DNA sequence from an initially small sample – sometimes even a single copy. It is a crucial process for a range of genetictechnologies and, in fact, has enabled the development of a suite of new technologies.on.
How does PCR work?
PCR mimics what happens in cells when DNA is copied (replicated) prior to cell division, but it is carried out in controlled conditions in a laboratory. The machine that is used is simply called a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put into the machine, and the machine changes the temperature to suit each step of the process.
Standard ingredients in the mixture are:
the DNA segment of interest
specific primers
heat-resistant DNA polymerase enzyme
the four different types of DNA nucleotides
the salts needed to create a suitable environment for the enzyme to act.
What is the PCR process?
Step 1: Denaturation
As in DNA replication, the two strands in the DNA double helix need to be separated.
The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation.
Step 2: Annealing
Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand.
Step 3: Extension
New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand.
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The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.
The cycle is repeated many times (usually 20–30) as most processes using PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies.
PCR technology is still developing. There is continuing development and refinement of the processes and tools used, allowing the process to beadapted to meet specialist needs. For instance, new methods and refinements are being developed and used, especially when quantification of DNA in a sample is needed. New methods include real-time PCR or quantitative PCR (qPCR) and digital PCR (dPCR). In qPCR, the amplification of DNA is monitored in real time, allowing the quantification of target DNA throughout the process. dPCR is a new, more refined approach that breaks the PCR process up into many smaller steps. It offers increased precision, more reliable measurements and absolute quantification from very small or mixed samples.
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