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INTRODUCTION: The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece

of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in !"# by $ary %ullis, &'( is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.. )n !!#, %ullis was awarded the Nobel &ri*e in 'hemistry along with %ichael +mith for his wor, on &'(. PROCEDURE: &'( consists of a series of -./0. repeated temperature changes, called cycles, with each cycle commonly consisting of -/# discrete temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (1!.2'), and followed by one hold at the end for final product e3tension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the en*yme used for DNA synthesis, the

concentration of divalent ions and dNT&s in the reaction. around 80 ' ( -!. ma. the polymerase attaches and starts copying the template. and the melting temperature (Tm) of the primers. the double/stranded DNA melts and opens into two pieces of single/stranded DNA.5<0 2' for 85 8 minutes after the .s complementary to the template are coupled to the primer.7).oined primer and template). )t is only required for DNA polymerases that require heat activation by hot/start &'(.s best.' ( 6 .ing a double stranded DNA molecule.. which is held for 5! minutes.. • Initialization step4 This step consists of heating the reaction to a temperature of !05!6 2' (or !" 2' if e3tremely thermostable polymerases are used). and DNA building bloc. • Denaturation: At !0 ' (-. • Extension: At <.) :n the small length of double/stranded DNA (the .7). the polymerase wor. .6 7). • inal elongation: This single step is occasionally performed at a temperature of <. the primers pair up (anneal) with the single/stranded 9template9 (The template is the sequence of DNA to be copied. • Annealing: At medium temperatures.

er). The si*e(s) of &'( products is determined by comparison with a DNA ladder (a molecular weight mar. run on the gel alongside the &'( products APP"ICATION: • The first application of &'( was for genetic testing. To chec. DNA samples for &renatal testing can be obtained by amniocentesis. or their children might be tested for actually being affected by a disease. &rospective parents can be tested for being genetic carriers. &'( analysis is also . where a sample of DNA is analy*ed for the presence of genetic disease mutations. chorionic villus sampling. which contains DNA fragments of .last &'( cycle to ensure that any remaining single/stranded DNA is fully e3tended.nown si*e. whether the &'( generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon). agarose gel electrophoresis is employed for si*e separation of the &'( products. • inal hol!: This step at 05 8 2' for an indefinite time may be employed for short/term storage of the reaction. or even by the analysis of rare fetal cells circulating in the mother=s bloodstream.

• Applications include DNA cloning for sequencing. where individual cells of a developing embryo are tested for mutations.. or 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 .". • %any forms of cancer involve alterations to oncogenes. As of -.essential to &reimplantation genetic diagnosis. DNA/ based phylogeny. vital to organ transplantation. there is even a proposal to replace the traditional antibody/based tests for blood type with &'(/based tests. therapy regimens can sometimes be individually customi*ed to a patient. >y using &'(/based tests to study these mutations. • &'( can also be used as part of a sensitive test for tissue typing.