Hi! Is it possible to amplify one gene starting from a mixture of different DNAs? I am asking because I want to determine the population of algae in some water samples through a metagenomic approach. But for Illumina sequencing I first have to produce a pool of amplicons with adapters.
So, let's say, if I have 10 different algal species in my sample, and I extract the DNA of all of them, I will have a mixture of 10 different DNAs. Then I want to amplify one gene of interest with specific primers, for all of the species. is this possible with a routine PCR?
The PCR products obtained will be then sequenced through Illumina and used for taxonomy classification.
Thank you in advance.
Hi Lorenza,
Yes, if you conduct DNA extraction in one combined sample you will have all DNA present.
You will be able to specifically amplify target areas in routine PCR provided that the PCR primers have been well designed.
In closely related species the genetic variation be very scarce so it can sometimes be hard to amplify specific product
If you have good data available for the species of algae you are looking at you can either design species specific PCR primers or a universal primer will amplify the gene across the various samples.
Another method though slightly more complicated is using a universal forward primer ( targeting a site conserved across all samples) and using species specific reverse primers.
The advantage of using this method is that if you can design the products of being various lengths you will know what species you have present without the need for sequencing and can be visualized on gel electrophoresis.
Hope this has been helpful.
PCR primers are able to amplify any matched DNA template in your sample. If the sequences to amplify are known, your can procede with an universal primers that can match with a conserved sequence in your DNA. so you have to perform a primers conception. if the conception is good, you primers can amplify 10 different templates.
I am using universal primers that amplify the plastid 23S rDNA gene, domain V. Do you think it should work? Of course then I will have (hopefully) ten different amplified fragments that overlap into the agarose gel (because the size of the fragment is almost the same in different species).
I agree with the idea of using a universal primer and a specific ones (maybe with a a specific tag/or as someone has said already designed to have different sized amplicons) in order to be able to differentiate the amplified fragments that you will obtain.If you don't need to differentiate them you can just use the universal primers.
Hope that helps
A big hug
Hello, as far as I understand it you will sequence the pool of amplicons and you do not need to pool the DNA extractions prior to amplification? if so you have two simple options : 1- modify your primers to incorporate barcodes at 5', run your 10 amplicons with different primer combinations (you need for example 4 forward modified primers + 3 modified reverse to make 10 combinations) and pool the amplicons in equimolar conditions. You can then ligate illumina adapters with standard protocol as in truseq nano kit for example. 2- an other option is to use a 1-step pcr involving your primers modified with a universal linker (5') and a different indexed adapter that will match the linker and add the illumina index and sequencing primers for each of your 10 amplicons. It is a bit more tricky but more universal as it requires only one type of primers and indexed adapters. The second method may require changing the miseq list of indexes what might be unpractical.
In any case you will be able to demultiplex your ten samples easyly after the run.
By the way those methods are highly scalable to hundreds of amplicons.
Good luck,
Jean-Francois
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