Documentos de Académico
Documentos de Profesional
Documentos de Cultura
amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
AMPLIFICATION
Amplification means making multiple identical copies
(replicates) of a DNA sequence. DNA amplification has proved very important in recombinant DNA technology and is used in a range of applications in medicine and forensic science. That method is PCR.
cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.
a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
PCR principle
PCR is used to amplify a specific region of a DNA
strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.
PCR procedure
Different steps are processed during thermal cycling
process while using PCR. Preparation of Reaction Mixture Reaction Mixture Set Up Components of the Reaction Mixture Cycling steps
mixture is prepared which contains buffer dNTPs primers and Taq DNA Polymerase.
aliquoted into individual tubes. MgCl2 and template DNA solutions are then added. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers.
0.01-1 ng Higher amounts of template DNA usually increase the yield of nonspecific PCR products
Primers.
primers are short dna sequences. During primer
selection diffrent guide lines should be kept in mind. PCR primers are usually 15-30 nucleotides in length. Longer primers provide higher specificity The GC content should be 40-60%. The C and G nucleotides should be distributed uniformly throughout of the primer..
complementary to any other primer in the reaction mixture, in order to avoid primer-dimer and hairpin formation All possible sites of complementarity between primers and the template DNA should be noted.
dNTPs.
The concentration of each dNTP in the reaction mixture is
usually 200 M. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level. When maximum fidelity of the PCR process is crucial, the final dNTP concentration should be 10-50 M, since the fidelity of DNA synthesis is maximal in this concentration range. In addition, the concentration of MgCl2 should be selected empirically, starting from 1 mM and increasing in 0.1 mM steps, until a sufficient yield of PCR product is obtained.
50 l of reaction mix. Higher Taq DNA Polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA Polymerase (2-3 u) may be necessary to obtain a better yield of amplification products.
magnesium concentration. Recombinant Taq polymerase This has the advantage over 'natural' Taq enzyme of greater batch conformity and, hence, higher reproducibility
Cycling Conditions
The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3 min at 95C if the GC content is 50% or less. This interval should be extended up to 10 min for GC-rich templates.
95C, Taq DNA Polymerase can be added into the initial reaction mixture. If longer initial denaturation or a higher temperature is necessary, Taq DNA Polymerase should be added only after the initial denaturation, as the stability of the enzyme dramatically decreases at temperatures over 95C.
Denaturation Step
Usually denaturation for 0.5-2 min at 94-95C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. If the amplified DNA has a very high GC content, denaturation time may be increased up to 3-4 min. Alternatively, additives facilitating DNA denaturation - glycerol (up to 10-15vol.%), DMSO (up to 10%) or formamide (up to 5%) - should be used. In the presence of such additives
. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1-2C.
Extending Step.
Usually the extending step is performed at 70-75C.
The rate of DNA synthesis by Taq DNA Polymerase is highest at this temperature. Recommended extending time is 1 min for the synthesis of PCR fragments up to 2 kb. When larger DNA fragments are amplified, the extending time is usually increased by 1 min for each 1000 bp.
TYPES OF PCR
AFLP PCR Allele-Specific PCR Assymetric PCR Inverse PCR In Situ PCR Long PCR Nested PCR RT-PCR or Reverse Transcriptase PCR Real Time PCR Single Cell PCR Standard PCR
Application of PCR
Isolation of genomic DNA
Amplification and quantification of DNA PCR in diagnosis of diseases And many more
discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.
DNA sequencing
applications of PCR include DNA sequencing to
determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing method.
targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a fortythousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a
amount of a given sequence present in a sample a technique often applied to quantitatively determine levels of gene expression
as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, viruses . The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
Thank you