Author Topic: The specificity of a PCR reaction?  (Read 12044 times)

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Offline Abdikasim

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The specificity of a PCR reaction?
« on: September 12, 2009, 07:10:31 AM »
What is the most critical step in determining the specificity of a PCR reaction??



Offline Mandeq

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Re: The specificity of a PCR reaction?
« Reply #1 on: September 14, 2009, 11:37:45 AM »
All the steps of PCR are very compulsory; I think DENATURATION OF DNA is the most critical or important step. Because with out DNA denaturation you can't determine DNA fragments.

Offline Abdikasim

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Re: The specificity of a PCR reaction?
« Reply #2 on: September 16, 2009, 08:51:44 AM »
it is annealing temperature. Annealing temp determines the amount of mismach tolerated between the primers and the target

Offline Mandeq

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Re: The specificity of a PCR reaction?
« Reply #3 on: September 16, 2009, 10:32:35 AM »
i think they both important , thnx 4 educating us brov

Offline Findhig

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Re: The specificity of a PCR reaction?
« Reply #4 on: September 17, 2009, 06:43:26 PM »

POLYMERASE CHAIN REACTION


[/b]In molecular biology, the polymerase chain reaction (PCR) is a technique to 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. The method relies on thermal cycling, consisting of 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. As PCR progresses, the DNA generated is itself used as 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.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1984 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These 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. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.[4]


Offline Anu Smith

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Re: The specificity of a PCR reaction?
« Reply #5 on: December 07, 2009, 03:30:14 PM »
Each and every step of PCR are very important. If any of the step is not performed properly or some mistake occurs then it is very difficult to get the result.

Offline Kasim

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Re: The specificity of a PCR reaction?
« Reply #6 on: June 30, 2011, 10:48:00 PM »
Brother Abdikasim is right...the annealing step is the most important one in PCR reaction.... this is because primers are very temperature specific and the amount of amplified product depends mainly on the efficiency of primers, their annealing temperature and time. then is the denaturation step.
Nice Discussion though
"Chance Favors Prepared Mind"

Offline Muruganloyola

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Re: The specificity of a PCR reaction?
« Reply #7 on: September 16, 2011, 03:47:39 PM »
As discussed by every one , In PCR most critical step is designing primer for specific target of expected genome or result, Primer designing includes various characteristics like, no primer dimer, primer should have more than 18 to 22 nucleotides, should rich in GC content, etc,


and another important step is optimising the exact annealing temperature of Primer with the target genome.

and optimising the reaction condition and the concentration of Primer in the reaction mixture are essential and important steps of PCR

I hope it would be useful for all..

thank you

ur

NMN

 

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