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Setting up a multiplex PCR assay. - (Feb/09/2009 )

Hello!

A novice is looking for some advice.

I need to develop a multiplex PCR assay to help ID 5 related insects. They belong to the same family. I want to use the mitochondrial Cytochrome C. Oxidase (C01) gene as my multiplex target.

I plan to amplify different lengths of the C01 unique to each insect. For example: 200bp insect 1, 375bp insect 2, 400bp insect three, ect. These different amplicon lengths will be measured on a gel to give me an assay for my particular insect.

I have aligned the 5 C01 insect genes in clustal. I plan to use the nonconserved regions of the alignment to develop unique primers for each insect. Here are some questions I have about this setup.

Would it be possible to use a universal reverse primer paired with 5 specific forward primers to amplify different lengths of DNA during PCR? Will that put too much stress on one primer?

How much difference should each primer have to make it specific enough for each insect.? The insects I am working with have similar C01 genes. Even at nonconserved sites my primers will share some common sequence information. I am willing to reject the C01 if my primers cannot be unique enough. Iím still in a planning stage.

Words of wisdom are greatly appreciated.

-genejock-

I am doing a similar setup designed to identify 17 different bacterial species from clinical samples using the 16s gene and the 16s-23s intergenic spacer region...

genejock on Feb 9 2009, 11:40 AM said:

Would it be possible to use a universal reverse primer paired with 5 specific forward primers to amplify different lengths of DNA during PCR?


Yes. In my setup, my universal primer is a forward primer selected from a section of the 16s gene conserved in all the species I'm detecting, and I have a reverse primer for each species that is specific for that species (chosen from the 16s-23s intergenic spacer region) and that produces a fragment size different from all others in the same multiplex assay. I had to do several multiplex panels, differing in the reverse (species-specific) primers used in the primer mix, because there's only so many unique and resolvable product sizes I can use, given the relatively small size of the intergenic spacer region.

genejock on Feb 9 2009, 11:40 AM said:

Will that put too much stress on one primer?


I haven't found this to be the case. I use twice as much universal primer in my mix as unique primers, though I'm not sure that's really needed.

genejock on Feb 9 2009, 11:40 AM said:

How much difference should each primer have to make it specific enough for each insect.? The insects I am working with have similar C01 genes. Even at nonconserved sites my primers will share some common sequence information. I am willing to reject the C01 if my primers cannot be unique enough. Iím still in a planning stage.


There is no absolute answer here. At a minimum, the primers should miss by several bases, preferably at the 3-prime end. I ran my multiplex mixes against panels of known isolates to ensure they generated a product only for the species they were intended to; several primers initially showed cross-reactivity and had to be tweaked, but I was ultimately successful at designing primers that produced a product only from the intended species.

Hope this helps!

-HomeBrew-

HomeBrew on Feb 9 2009, 09:08 AM said:

I am doing a similar setup designed to identify 17 different bacterial species from clinical samples using the 16s gene and the 16s-23s intergenic spacer region...

genejock on Feb 9 2009, 11:40 AM said:

Would it be possible to use a universal reverse primer paired with 5 specific forward primers to amplify different lengths of DNA during PCR?


Yes. In my setup, my universal primer is a forward primer selected from a section of the 16s gene conserved in all the species I'm detecting, and I have a reverse primer for each species that is specific for that species (chosen from the 16s-23s intergenic spacer region) and that produces a fragment size different from all others in the same multiplex assay. I had to do several multiplex panels, differing in the reverse (species-specific) primers used in the primer mix, because there's only so many unique and resolvable product sizes I can use, given the relatively small size of the intergenic spacer region.

genejock on Feb 9 2009, 11:40 AM said:

Will that put too much stress on one primer?


I haven't found this to be the case. I use twice as much universal primer in my mix as unique primers, though I'm not sure that's really needed.

genejock on Feb 9 2009, 11:40 AM said:

How much difference should each primer have to make it specific enough for each insect.? The insects I am working with have similar C01 genes. Even at nonconserved sites my primers will share some common sequence information. I am willing to reject the C01 if my primers cannot be unique enough. Iím still in a planning stage.


There is no absolute answer here. At a minimum, the primers should miss by several bases, preferably at the 3-prime end. I ran my multiplex mixes against panels of known isolates to ensure they generated a product only for the species they were intended to; several primers initially showed cross-reactivity and had to be tweaked, but I was ultimately successful at designing primers that produced a product only from the intended species.

Hope this helps!


Homebrew

This is good information. Thank your for your input. I'll continue to design the assay off of the C01 and see where it goes.

Best regards

-genejock-