When can we use them ? Why after a multiplex PCR sequencing is not carried ?
Hi, maybe you should read some publication or buy a book where the different PCR techniques are explained. Cheers, Nadine
Hi Meriem,
They're really just variations of a theme depending on your exact research question. Nested PCR is a technique used when your primers display non-specific binding. Wikipedia has a nice diagram of how it works, which I've linked below.
Multiplex PCR uses multiple primer sets in a single reaction, targeting different genes (or reporter regions). This is useful in diagnostic settings where combinations of virulence factors can be used to identify a pathogen, particularly if you're trying to identify a strain or complex. I haven't really through about why you couldn't sequence from a multiplex PCR directly - I suppose it's because you have a lot a non-target DNA in your solution which makes it difficult to quantify your template, and there's always the risk you'll get non-specific binding when you sequence. You could easily gel-extract your fragments then sequence the DNA if needed, though.
Quantitative PCR is pretty much what the name suggests. It allows you to measure the accumulation of your product as the reaction progresses. This allows you to measure the abundance of template DNA (or cDNA) in the reaction which is useful when looking at the expression levels of genes or, as is common in microbiology, quantifying how much of the total bacterial community a particular species represents. qPCR is also popular in diagnostic work because the reactions are much faster to perform that a standard PCR and you don't need to run the product in an agarose gel to see the result.
http://en.wikipedia.org/wiki/Nested_polymerase_chain_reaction
Article Diagnostic multiplex PCR for toxin genotype of Clostridium p...
Hi, each method has its own advantage
by passing time, newer methods have been discovered. As a result more ways utilized, and of course most scientists have the significant goal to compare the results of different methods.
Regards
Dear Merieum,
i would like to give you simple answers directly to your query.
Uniplex PCR- Specific targeting single gene or loci. (Only one forward and reverse primer)
Nested PCR- Highly specific and sensitive to gene target, which amplify a target sequence (for eg gene length of 1000bp --- Forward primer starts at 25bp and reverse primer binds to 950bp, single round, (Here you will add DNA from the extraction from any specimen or culture) Product size will be 925bp here.
But in the second round you have to take 1-5 microlitre of PCR product as a template for Nested PCR reaction.
and in the second round, set 2 forward primer binds inner region at 200bp and reverse primer binds to 700bp ) so the product size would be 500bp here.
so a same gene getting highly specific product of different band size.
Multiple PCRs carrying more than one pcr products, carrying multiple bands,
Hence, direct sequencing from Multiplex will yield bad sequencing result.
You can elute the Specific product from the gel of multiplex product and purify it , then you can go for sequencing,
For further details read some molecular books mentioned above.
Cheers,
NMN
Hi
Multiple Template PCR Reaction: It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.
Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first one.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously detect or quantify a targeted DNA molecule. This method is too sensible.
Hi, I agree with above nice discussions.
these techniques are based on different principles, different purpose and applications.
Regards,
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