Limit of detection for salmon gDNA? - (Jul/29/2013 )
I'm trying to detect very small amount of salmon DNA. I'm using qpcr with primers for two different mitochondrial genes, Cyto oxidase subunit 1 and Cyto b. I'm hitting my limit of detection around 1pg. I'd like to increase it but I'm not sure how. Part of my problem is that I'm not sure how many copies of my genes are present in 1pg of material. I have tried two primer sets for each gene and used both probe detection and two kinds of Sybrgreen.
Any advice? Should I switch genes? Do pre-amplification?
I'd say it depends on the source of material as some tissues and their cells respectively, contain more mitochondria than others e.g. liver cells. One first idea would be to try to maximise the DNA yield during isolation....
Mitochondrial genes generally are a good idea as usually few to very many mitochondria are in all cells and they contain several DNA copies too.
This is salmon testes DNA. I haven't been able to find any papers that could say how many copies of mitochondrial DNA would be present in that tissue type. Our DNA is good quality but we want to be able to detect very small amounts of it.
How much copies you have I've no idea, but if PCR is working (?) you can do dilutions of course from this working samples and see what amplification you get at what dilution. The DNA amount of the stock you can measure before with a photometer (for example). Even if this may not me very exact you get an idea of the sensitivity of the reaction.
Yes, I've done a dilution series but I'm not getting past detection past 1pg per reaction. My effeciencies are a little low (around 87%) but I've tried four different primer sets and I haven't been able to improve that.
Well 1 pg sounds really good for me (I work usually with nanograms) and I wonder if you really can go below a certain threshold. Other ideas would be lots of optimisation with Mg2+ and dNTP concentrations, cycle numbers and all that (if you didn't do it already) and surely pre-amplification is an option...
Finally it's also a question if you get a stable amplification with all samples, when having so low template amounts, you might then have a problem of false negatives...
And have a look here: https://www.youtube.com/watch?v=YU8vX3UGqKw
Better yet, check out. Www.bio-rad.com/supermixes_tutorial. It teaches you about how Taq polymerase is less sensitive and how to validate your work.
AFAIK theoretical limit of PCR is one copy of template. Real-time PCR with primers of good efficiency should be able to detect such limit.
Problem lies elsewhere, if the template is diluted enough, you have problems with uneven spliting of sample when pipetting (small numbers probem).
If the template is more complex, ie. you are detecting low abundant template inside genomic DNA, it's also a problem.
To test your primers detection limit, you can make an artificial control either by cloning the PCR product of your primers into plasmid (better way), or just amplifing much of it and purifing it. You can then calculate copy number of these controls and dilute to see how many copies are your primers able to detect in ideal conditions.
From there you can assume if the primers are the weak link of your reaction.