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Scientific Technique Molecular Biology Amplify DNA DNA Sequence Kary Mullis

PCR is a technique used to amplify a small amount of DNA across multiple orders of magnitude. It involves repeated temperature changes called cycles with three steps - denaturation to separate DNA strands, annealing where primers attach to strands, and extension where DNA polymerase builds new strands. Each cycle doubles the amount of DNA. PCR is used for genetic testing, cancer treatment, forensics, evolutionary studies, and more. It has become indispensable for medical and biological research.

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97 views4 pages

Scientific Technique Molecular Biology Amplify DNA DNA Sequence Kary Mullis

PCR is a technique used to amplify a small amount of DNA across multiple orders of magnitude. It involves repeated temperature changes called cycles with three steps - denaturation to separate DNA strands, annealing where primers attach to strands, and extension where DNA polymerase builds new strands. Each cycle doubles the amount of DNA. PCR is used for genetic testing, cancer treatment, forensics, evolutionary studies, and more. It has become indispensable for medical and biological research.

Uploaded by

Sai Sridhar
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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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, and the melting temperature (Tm) of the primers.

Initialization step4 This step consists of heating the


reaction to a temperature of !05!6 2' (or !" 2' if e3tremely thermostable polymerases are used), which is held for 5! minutes. )t is only required for DNA polymerases that require heat activation by hot/start &'(.

Denaturation: At !0 ' (-. .- 7), the double/stranded DNA melts and opens into two pieces of single/stranded DNA. Annealing: At medium temperatures, around 80 ' ( -!.- 7), the primers pair up (anneal) with the single/stranded 9template9 (The template is the sequence of DNA to be copied.) :n the small length of double/stranded DNA (the ;oined primer and template), the polymerase attaches and starts copying the template. Extension: At <- ' ( 6 .6 7), the polymerase wor,s best, and DNA building bloc,s complementary to the template are coupled to the primer, ma,ing a double stranded DNA molecule.

inal elongation: This single step is occasionally performed


at a temperature of <.5<0 2' for 85 8 minutes after the

last &'( cycle to ensure that any remaining single/stranded DNA is fully e3tended.

inal hol!: This step at 05 8 2' for an indefinite time may


be employed for short/term storage of the reaction.

To chec, 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. The si*e(s) of &'( products is determined by comparison with a DNA ladder (a molecular weight mar,er), which contains DNA fragments of ,nown si*e, run on the gel alongside the &'( products APP"ICATION:

The first application of &'( was for genetic testing, where a sample of DNA is analy*ed for the presence of genetic disease mutations. &rospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. DNA samples for &renatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother=s bloodstream. &'( analysis is also

essential to &reimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.

&'( can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of -..", there is even a proposal to replace the traditional antibody/based tests for blood type with &'(/based tests.

%any forms of cancer involve alterations to oncogenes. >y using &'(/based tests to study these mutations, therapy regimens can sometimes be individually customi*ed to a patient.

Applications include DNA cloning for sequencing, DNA/ based phylogeny, 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

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