PCR Fundamentals





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Published on Jul 9, 2012

For more information, visit http://www.bio-rad.com/yt/1/PCRtechno....
This video describes the fundamental principles and procedures of the polymerase chain reaction (PCR), which has become an essential tool for molecular biology since its invention by Kary Mullis in 1983. PCR amplifies a DNA sequence of interest by making many copies of the target sequence, in a process similar to that used by cells during replication. The process consists of three basic steps that occur at different temperatures:

Denaturation — double-stranded DNA fragments are heated to a temperature near boiling to split them into single strands.

Annealing — the reaction is cooled to allow small oligonucleotides with specifically designed sequences, called primers, to bind with the single-stranded DNA at locations where their sequences are complementary. This creation of short segments of double-stranded DNA "primes" the target sequence for replication by DNA polymerase, which associates with the ends of these segments.

Extension — at a third temperature, the DNA polymerase extends the short primers, filling in bases complementary to the target sequence by adding dNTPs that are present in solution. Finally, the complete double-stranded target sequence is regenerated, thus doubling the number of copies present in the original sample.

These steps are repeated a number of times in an instrument called a thermal cycler until a sufficient number of DNA copies have been generated. Because the number of copies of the target DNA is doubled in each cycle of three steps, this number grows exponentially in a chain reaction.

Since its introduction, subsequent development of advanced PCR techniques has revolutionized molecular biology by enabling rapid cloning, gene sequencing, mutation analysis, genetic recombination, DNA and RNA quantitation, and other breakthroughs. When asked what uses he envisioned for PCR, Mullis once quipped "PCR is to DNA what the screwdriver is to screws". Today, there is a PCR machine in every DNA lab.


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