Yes that makes sense. Separating eg 70 from 74 looks much easier and run the gel slowly and cold to minimise diffusion. You might in this type of amplification find that in the last pcr cycle when you have a heterozygous sample find that a slow moving third band appears which will be the heteroduplex of small and larger which will also be indicative of a heterozygous sample

Hi,

Your solution is capillary electrophoresis. Try to find it from your colleagues. We use Qiaxcel with High Resolution DNA kit. It distinguishes even 2bp difference.

Good luck

It would be easier to use a high percentage metaphor agarose type of gel. These can be up to 4% agarose and routinely separate small fragments. If you use acrylamide 10% is a bit high, try 4-6%.

I agree with Gregory Dressler that agarose is usable but if the 2 fragments are variant of the same sequence then I would try to find a common restriction site between the 2 sequences . If you could cut the fragments so that there was one common fragment and then you are separating ,for instance, 100 from 104 bases then the difference would be more obvious and the separation better. Run the gel in a cold room and at half your usual voltage to have less thermal diffusion of the bands so better separation

General rule of thumb is that you can distinguish products that are 10% or more different in length with standard agarose electrophoresis.

But you may as well try it and see if it works. You'll want to use the largest gel you can pour to allow as much physical distance for separation as possible. Run the gels "low and cold" (low voltage, in a cold room). Include a full set of controls and a size ladder. Check on it often to make sure the bands don't run off the end.

Unless you NEED to recover the products from the gel, it might be easier and cheaper to either digest or sequence instead.

You might want to consider acrylamide instead.

I used to separate amplicons with differences of 2bp on vertical PAGE. But theoretically, I do agree with Katie which separation below 10% differences between amplicons would be a great challenge on normal agarose gel unless you make METAPHOR agarose which will help you to have a resolution of 2bp.

Teitur Sævarsson < amplify and analyze two distinct dsDNA fragments , one being 238 bp in size and the other 242 bp. >

You meant you used 1 pair of primers and you can amplify both these two 'distinct' dsDNA fragment in a PCR tube, and now you try to separate them? Or 2 pairs of primers, and you just want to see if they are different (4 bp), running them on gel side by side?

You can tri with metaphore gel from Takara, Japan with 5% concentration, which I have found can separate even 3 bp in normal electrophoresis system rather than Qiaxel from Qiagen.

Yuan-Yeu Yau

I'm essentially using 2 pairs of primers simultaneously in the same PCR tube, and need to see if they are different. I'm doing a junction point and 5' breakpoint assay to see if a certain duplication is present at the respective locus. So I'm running the two primer pairs together on each gDNA sample and need to be able to separate the two fragments to be able to tell whether or not said individuals carry a duplicated copy or not.

I would opt for capillary electrophoresis for this procedure but given my limited resources at this point I'm forced to take different measures. Hence my inquiry.

Can you move the primers closer to give a smaller pcr product so a greater percentage difference for the two amplimers?

Paul Rutland

I might be able to, or at least something of the sort. The primers I'm using were designed in another similar experiment by Norris & Whan (2008) and have been used by several other researchers since, with adequate results. So, I admit, I didn't give it much thought before venturing onward. I might simply be able to move or redesign the 5' breakpoint primer pair, increasing the percentage difference by either slightly enlarging or diminishing one of the PCR products. Does that make sense?

Yes that makes sense. Separating eg 70 from 74 looks much easier and run the gel slowly and cold to minimise diffusion. You might in this type of amplification find that in the last pcr cycle when you have a heterozygous sample find that a slow moving third band appears which will be the heteroduplex of small and larger which will also be indicative of a heterozygous sample

Teitur Sævarsson Thanks for answering my questions. I checked the prices of kits mentioned above (see below). There are already several people recommending using Metaphore gel. You should probably try both your shorter amplicons (from your new designer primers) (suggested by Paul Rutland ) and longer amplicons (from your original primers) on Metaphore gel.

1. Qiaxcel with High Resolution DNA kit --> $1,079

https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaxcel-dna-kits/#orderinginformation

2. Metaphore Gel (25 grams) --> $202.24

https://us.vwr.com/store/product/20545309/metaphor-agarose-lonza#order

Please note that Qiagen kits are for 1200 reactions. Cheers

On increasing the % of gel one should be able to resolve two fragments of even 4 bp difference.

Try to prepare 40% native PAGE for DNA using TAE buffer.

Refer Shambrook for preparing native PAGE.

Instead of mini gel try using midi or large gel. (The separation of fragments would be better for the kind of difference you are interested in). Try to run the gel in cold conditions with 150-200 V.

You may stain the gel with Ethidium Bromide or use silver staining procedure for native gel.

In any case you must use homogeneous size primers (gel purified grade) that migrate 100% in a single band on a denaturing small >16% urea PAGE. Better to load also a non gel purified oligos on the same gel as a control. Simply HPLC purified primers usually display satellite N-1, N-2 bands. I do not trust commercially gel purified oligos. We command HPLC purified grade primers and do ourself the purification on appropriate single nt resolution urea PAGE.

If you are in an old lab that used to run sequencing gels, just do that. For a refresher, see this Article Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Not necessarily this is a question of gel! Try temp adjustment and see if it works

Dear Teitur,

What's the objective of the PCR assay and how does the 4 bp difference distribute in the fragment that you are amplifying?

If your goal is to genotype the locus, and if the 4 bp difference is not due to unit number difference of some repetitive sequence, you probably can design two allele-specific primers targeting the polymorphism. The drawback is that these are dominant markers, and now you need to do two PCR reactions instead of one to detect heterozygosity. But running the gel and data interpretation will be much more straightforward. So there are trade-offs.

Another thing that you can try is to see if the 4 bp difference introduces some restriction sites that are unique in one of the two alleles. If there are, then you can do a simple enzyme digestion of the PCR product and run them out on a regular gel. These should be co-dominant markers that allow you to differentiate between homozygous and heterozygous.

Hope it helps.

All best,

Sheng

You could use fluorescent primers and detect the PCR-products on an automatic sequencer. The resolution is even higher than 1 bp.

Thank you for your detailed and helpful response, Sheng Sun

The main goal is to visually distinguish between the two fragments post electrophoresis, as the presence of both fragments in the same sample confirms the presence of duplicated copy(ies) in the respective individual. Currently, this confirmation is the only thing we are seeking, genotyping the locus is not a main priority, at least not yet. We are using pre-designed primers as I believe mentioned in one of my earlier responses in this discussion, and they appear to be working, but the problem is that I'm not able to accurately distinguish between the fragments with my current method of electrophoresis.

I might check for restriction sites within one of the fragments, or as Mr. Rutland mentioned, redesign on of the pairs. However, I've been having trouble finding the sequenced duplicated locus in the genomic databases, even through the initial study of the matter. Because of that, I've been unable to examine one of the fragments (the junction between the original gene and the duplicated copy) in the genome browser.

After reviewing the suggestions in this discussion and consulting with my professor, I'm going to try using polyacrylamide gel to see if it suffices to separate the fragments efficiently, as well as looking into possible restriction sites within the fragment I'm able to examine.

I'd like to use the opportunity and thank all of you for your responses to my inquiry. They've all been helpful and quite educating. To an inexperienced aspirant, it means a lot!

I'll be sure to post an update on the results when the time comes.

My best regards to all of you,

Teitur

in old days, people use to use PAGE to sequence at single nt levels. I guess you can use PAGE ,

Of course, PAGE is suitable. It has a comparable resolution as automatic sequencers but will be much more work. Additionally, you need to visualize the bands using either silver staining (requires some experience), radionucleotides (requires a dark room) or any other method.

If your institute has the 'ABI PRISM® 3700 DNA Analyzer' facility that is the best and even you can differentiate single base pair difference. As per my knowledge University if Iceland has ABI facility, I did some sequencing when I was there. You can contact them and carry on your work. You can use that machine for 'Genomics and other Large-Scale DNA Analysis Applications' too. As mentioned many PAGE is other best option. Good Luck!

Yes, old style PAGE will resolve 4 bp easily, but, as said above, you need a good detection system.

You can use PAGE, but it requires much work

You can resolve DNA by 4 bp using 8% native PAGE.

PAGE (Polyacrylamide Gel Electrophoresis). It's run vertically unlike the horizontally run agarose gel. So it is of higher resolution up to the nucleotide level.

You can do it quite easily with PAGE - the key is to use relatively high gel concentration (10-12%), run it very, very... very slow (low current), and keep it cold for the entire time - ice it and change the ice or ice packs often. Good luck!