I agree that shearing of DNA should not influence the UV measurement. I hope I don't miss the point here but it might influence your qPCR since the shearing will disrupt a fraction of the PCR template! Or in other words: The same amount of unsheared/intact template DNA will give a higher result (lower Ct) in qPCR than randomly sheared DNA. Or to answer with your words: The UV measurement of sheared DNA overestimates the true amount of available template DNA for the qPCR!

Are you using same kit for extracting DNA of your samples and P. aeruginosa (which you are going to use as a standard)? If, yes, then don't worry. If you got your DNA in different methods, then it will affect your curve. Whatever, just run one experiment with your standard and draw a curve and see how it look like. For the estimation of DNA, which instrument did you use? Nano drop? Qubit kit? If you have values of 280/260 and 260/280 you will understand better the reason of shearing. Also, which kit and which elution buffer did you use in extraction process. Did you treat your sample by Rnase at some point? In my experience, shearing would not affect your qPCR quantification as I have extracted DNA of samples and positive control with the same kit.

Hi Maria

The shearing in it self should not affect UV measurements of DNA. However, the level of shearing does have other implications which should be considered; primarily that it can be extremely difficult to accurately pipette small volumes (< 5 ul) of high molecular weight DNA as the DNA tend to tangle. Therefore, a certain amount of shearing is probably desirably. It follows from this, that it can be difficult to accurately measure DNA concentration of high molecular weight DNA on a NanoDrop. I am not sure how shearing affects dye based measurements but I imagine that the above considerations should apply as well..

Here is a link to a paper that you might find interesting

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576356/

Good luck

Jesper

I agree that shearing of DNA should not influence the UV measurement. I hope I don't miss the point here but it might influence your qPCR since the shearing will disrupt a fraction of the PCR template! Or in other words: The same amount of unsheared/intact template DNA will give a higher result (lower Ct) in qPCR than randomly sheared DNA. Or to answer with your words: The UV measurement of sheared DNA overestimates the true amount of available template DNA for the qPCR!

I used different kits for extracting soil and bacterial DNA. In fact, I think using the same for Pseudomonas would cause more shearing, as the kit for soil has micro beads and a heavy vortexing step to disrupt soil particles, detach cells, and break membranes.

Thanks Jesper for the article! should be very informative

While shearing is not so bad in my DNA sample (size range >10000- 500 bp), Tobias is right according to his reasoning and the paper kindly provided by Jesper.

So, I'll try a new DNA extraction and see how it works.

This only would happen if your template is very large. Normal handling of your DNA would lead to fragments larger than 1-2 kb

shearing DNA could result from DNA processing and/or bad bacterial culture. Make sure to start with a fresh culture.

Cees, the amplicon size is about 150 bp. Thus, do you consider that the fragmented DNA template would not be a problem for qPCR?

Hi Maria,

The shared DNA should not affect the measurement of DNA concentration or qPCR generally. However, RNA can affect both. It is worth checking if you RNAase treatment was sufficient.

Yes sheared DNA does increase the UV absorption and it could lead to overestimation of the concentration, but then it if you are having some experience with DNA estimation at 260nm, you will realize this at once. Of course the increase in absorption will be proportional to the extent shearing has occurred. Normally do not use such samples of DNA if the shearing is more than 3% (though some people still find themselves comfortable with shearing as high as 10%). Its up to you. Now my question is : HOW DID THE SHEARING OCCUR?

The vast majority of the advice given has been useful. So, to sum up U.V absorption is not affected by the level of shearing, as U.V absorption is not dependent on the "intactness" of DNA. qPCR a of a 150bp amplicon will only have a drop in efficiency when shearing has produced very short fragments of DNA. If there is any inhibition of the PCR, due to RNA, this would be seen in the efficiency value being

Useful summary! Thanks Malcom. Will run my qPCR and evaluate the efficiency to decide whether it' s necessary to try an improved DNA extraction

María,

1- No, shearing does not change detectably the absorption coefficient of ds-DNA (which depends on base composition/stacking rather than length).

2- Unless you are dealing with samples derived from agarose-embedded bacteria digested in situ, all samples of genomic DNA you have come accross and will come accross are sheared to one extent or another. The point is not whether shearing influences qPCR amplification (it does), but when does it start to affect it noticeably. That in turn depends mainly on amplicon size (the smaller the better) and degree of fragmentation (idem). If you look at the paper Jensen above linked to, you will notice that shearing becomes a significant problem only once it gets down to a median size of 150 bp (their amplicon is 125 bp long).

I'd say, if the median size of your DNA is above 5 kb, go for it. Otherwise, repeat the genomic DNA prep. It should be possible to derive the necessary median size to amplify 150 bp with, say, 99% probability, by using Poisson statistics.

Hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level.

I realise there may be some confusion between denatured DNA i.e; two stranded DNA becoming a single strand either by heat or increasing alkalinity and DNA that is sheared to produce fragmented DNA. As Apurva correctly points out, hyperchromicity does occur during denaturation, but I was referring to fragmented DNA.

Basically no, it would not affect your U.V reading but interms of the DNA template to be quantified, you will have a problem with junk results as you will not be able to amplify the target sized template. your RNA/ DNA quantification ratio should tell you how good your RNA or DNA is ... otherwise load a small quantity of either on agarose gel and check for integrity visually...

No it shouldn't affect the concentration measured. Also for your qPCR you're safe as long as you normalize to a housekeeping gene since shearing will occur randomly the housekeeping gene should be similar affected than your target gene. So i do not agree with a previous comment that shearing could affect you qPCR results. At least not if you normalize every sample in a similar way.

I think use of sheared DNA is not recommended and should not be used for any DNA work be it PCR/qPCRThe junk along with normal DNA would also get amplifiedso actual target sized template may be misleading and can be missed.Logically this is the reason why we measure the DNA/RNA ratio and judge the quality of good DNA

>>>>>>Would sheared DNA increase UV absorption lead to overestimation of the concentration, thus affecting the standard curve?

....if you have a lot of RNA it would. I would use Pico Green assay to determine DNA concentration. Also don't forget to vortex your reagents before QPCR (it matters)