I need help with PCR problems - (Dec/21/2014 )

HI all I am very new to the whole PCR and lab scene. I am an undergrad student doing a scholarship and have NO idea what is going wrong with my PCR.

 

Long story, I'm sorry.

During the semester, I designed 5 pairs of primers for 5 different gene regions for 2 species. These primers came and I attempted to optimise them doing temperatures and cycles. One was happy doing amplification no matter what conditions I put it through (I like that one. That was a nice one), some took a bit of work, and most took a very long time to get right, with few not doing a thing.

 

At the end, four pairs worked, one did not. For sequencing, only two gave quality reads which were reliable. These were thrown into GenBank to check I amplified the correct region and sure enough, there were hits.  I was excited. However, this was for one species, nothing worked on the other. (Species A worked, Species B did not.)

 

However, for the scholarchip, I attempted to use the same two primer pairs that worked on Species A on Species B, as B was their target species. It did not work no matter how much I tweaked things. Blank after blank. Even attempting the positive control on Species A did not yield results at all. So I started to attempt to optimise again - I changed out all the reagents exept the primers and it still didn't work. I created a new working solution from my concentrated stock of primers and even with all new everything else, it didn't amplift anything. And yet it worked during semester? I used the exact same program and even the exact same Eppendorf machined in the lab.

^ Problem 1: Previously working primers aren't working. Even the one that worked no matter what previously. Almost worked TOO well. Had to lower the cycle number for that one.

 

So I gave up and attempted to work on the three regions that did not work during semester. I had designed new primers and they arrived. Optimisations for one pair showed some non-specific binding (which was expected, as that region is very odd in the sequence I was given to work with and has never specifically bound, but the most intense band is definitely the region I am after) but the other two pairs did not bind at all. This was a temperature gradient.

I decided to ignore these two for the moment, kinda really wanting results. So I concentrated on the region that did work. I upped the temperature as there was less non-specific binding at higher temperatures, and increased cycle number. Solid bands of non specific binding occurred again, but that's fine - I figured I could always gel purify the region I desire.

However, when I put the primers to the test on Species B, they did not work. They're not targeted for Species B, but my supervisor seems keen on these primers sequencing the same regions on the two species.

My issue was that Species B has NEVER worked. So I took it to him and he agreed to let me test the quality of the DNA using CO1 barconding. All three samples (Species A cDNA, Species A DNA and Species C DNA) DID amplify, so I concluded that the quality of the samples is okay.

^ Problem 2: Nothing working for Species A, replaced everything, still not working, even new primers.

 

I have run out of ideas short of introns getting in the way (all optimisations are done on cDNA, as DNA is very limited) - however my supervisor has said that he doesn't see how introns would affect things.

 

So I'm at a loss. I've tried replacing all my reagents, I've changed temperatures and cycle number, and I've tested the quality of the samples I am using as templates.

I don't understand why most of the time there is no binding on regions where they used to bind.

 

My supervisor suggested mix-and-matching the primer pairs for the three gene regions which did not sequence during semester. So I mixed the Old Upper and New Lower, and then the New Upper and Old Lower. I honestly don't know why he said to do that other than to hope for the best, as they have different TM's and I cannot imagine there being much happiness between the two if they want different optimal annealing temperatures.

I did not optimise for the mix-match, only attempted using a PCR program that kinda worked during the semester, tweaking it a bit to fit what the company that synthesised my primers told me the TM was, and the program used to design them told me the the rough optimal annealing temperature for the two different ones.

 

 

All and any help will be greatly appreciated.

Thank-you all for your time.

There's a lot here, so I might have missed some things in this reply:

 

How did you design the primers? Did you design based on the cDNA sequence for the gene or the genomic sequence? Did you design degenerate primers?

 

The ones that worked previously and are not now - have you tried ordering fresh primers of the same sequence? Often stocks of primers will get degraded with repeated freeze/thaw cycles and stop working. It could also be the template - this will degrade too over time and is dependent on storage.

 

If you designed your primers based on cDNA sequence and are amplifying off genomic sequence, then introns can play a role - they could be too big to effectively amplify across. If you are working off cDNA and designed off genomic your primers might lie halfway across an intron/exon boundary and not have sufficient binding to give a specific product.

 

Have you tried secondary structure inhibitors for those amplifications which don't work, such as betaine or DMSO?

Hi Bob1, thanks for the reply.

I was using the Oligo software. I cannot recall the version off the top of my head - it's in my lab book in the office where I am not, sorry.

The sequences I designed the primers off of were supplied by a researchers, I believe it was cDNA as of my initial two that worked, I found intron regions when I aligned it to their sequence.

I do not know what a degenerate primer is, so I did not design it intentionally.

 

I have not tried ordering new primers of the same sequence - and the original stock was only thawed initially to create a working stock when I got it. I've now thawed some twice to make a second working solution. But I do not think repeated freeze-thaw cycles would do it, unless someone else in the lab (communal) was leaving the primer box out for some very silly reason.

 

I tested the template by doing the CO1 amplification, which succeeded. According to my supervisors, this means that the templates are fine.

 

I have thought of there being introns getting in the way, however I haven't much control over that. My supervisors aren't really keen on doing too much more on this project, they wish me to move to a newer project for them. If everything else works, could this be the sole reason behind them not working? I'm almost at the point where I just want to know WHY i've been failing.

But then this doens't explain why my previously working PCR is now not.

 

 

I have not tried adding any inhibitors of secondary structures. I didn't know they existed either. I could take it to my supervisors and see how they feel about attempting to add this to the protocol.

Some things to try:

As Bob1 stated, you could add 5% of a betaine solution, which can make high GC content PCR work when it fails otherwise.

If you don't know about introns, then you could perhaps amplify the cDNA, or make new cDNA and work with that.

I would check the primer sequences with the tools at idtdna.com. Specifically, I would check the homodimer and heterodimer structures. Ones that show strong binding, especially at the 3' end, are bad. Particularly of concern are the ones which bind and allow extension of the 3' end of a primer. Binding in other places is not so important.

What is the expected GC content of the organism you are working with?

Degenerate primers are those designed to amplify many different species even if the species differ in the primer binding sequence. For instance if you have species A with sequence ATGATG and species b with ATCATG then for a degenerate primer you would order ATSATG which would basically make a mix of primers with the sequences for species A and B in the same tube.

 

If you didn't do this - how different are the primer binding sites between each species?

 

The fact that your previously working PCR is not now working implies that something has stopped working, this is most likely one of the degradable products: nucleotides, primers, polymerase or template DNA. As you have changed everything else (note that stocks fail sometimes too) the most likely is the primers.

Phage: Wow that site has amazing tools! Thank-you so much for showing that to me. I have found a lot of weak binding in one primer pair, and one particularly strong primer dimer at the 3' end of one. I'll definitely use this tool on the remainder of my primer pairs to see if this occurs more frequently than not (and most definitely reference it when designing new ones). The program I used only shows a small subset of self-binding dimers, whereas this is showing quite a lot of possibilities.

The GC content of the target regions depends on the regions. I'm expecting the lowest to have 35.2% and the highest to have 42.3%  (according to the program I used to help design the primers).

I'll try asking the supervisors about possibly adding the 5% betadine solution. I haven't had an opportunity since they've all been on leave.

 

 

Bob: I did not know this was an option to be had. It's definitely a good option. I have been designing my primers only on one species (target species with a mutation / region of particular interest) and trying to amplify the regions on alternate species. These species though related are highly divergent from the ancestral species and state. These gene regions of interest are particularly bad as they've undergone massive selection / lack of selective pressures (in theory), depending on the species under same conditions. So I'm afraid that using this degenerate primers as a possibility may not work either in practice or due to costs.

I did try to design the primers in regions of almost homology between species, and once I designed based on a consensus sequence of all (noob mistake), but there's still variation across species, and not always consistent variation.

 

I suspected it was the primers, it's unfortunate as I loathe to spend much more of their research money for this scholarship - which isn't giving much positive results.

 

 

 

Thank-you both so much for your input :) I am greatly appreciative of your help.