Water for PCR
Application Overview
High Purity Water for PCR
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from DNA polymerase. PCR is used to amplify a piece of DNA by in vitro enzymatic replication. It uses repeated cycles, each of which consists of three steps:- Step 1:
The reaction solution containing DNA molecules (to be copied), polymerases (which copy the DNA), primers (which serve as starting DNA) and nucleotides (which are attached to the primers) is heated to 95°C. This causes the two complementary strands to separate, a process known as denaturing or melting.
- Step 2:
Lowering the temperature to 55°C causes the primers to bind to the DNA, a process known as hybridization or annealing. The resulting bonds are stable only if the primer and DNA segment are complementary, i.e. if the base pairs of the primer and DNA segment match. The polymerases then begin to attach additional complementary nucleotides at these sites, thus strengthening the bonding between the primers and the DNA.
- Step 3:
Extension: The temperature is again increased, this time to 72°C. This is the ideal working temperature for the polymerases used, which add further nucleotides to the developing DNA strand. At the same time, any loose bonds that have formed between the primers and DNA segments that are not fully complementary are broken. Each time these three steps are repeated the number of copied DNA molecules doubles. After 20 cycles about a million molecules are cloned from a single segment of doublestranded DNA.
As PCR advances, the DNA generated is 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.
Developed in the 80s by Kary Mullis, PCR is now a common a cornerstone technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.
Simple qualitative PCR has evolved to quantitative PCR, allowing monitoring in real-time the presence and the amplification of DNA sequences. The principle is based on the use of fluorescent chromophores attached to the primer, and released upon elongation of the new DNA strand by the DNA polymerse.
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