Another approach to DNA quantification

[Simon Quellen Field]

We have been discussing using absorption of UV by a cuvette as a way

of finding out how much DNA is in a sample.

But there is a much simpler (cheaper) approach to DNA quantification in

use that is very DIY hackable, and applicable to 10 microliter sample sizes.

Simply measure the capacitance of the sample.

Measuring capacitance with a microcontroller is very simple.

Two wires, or two traces on a PC board make the capacitor.

The dielectric is the sample placed across the wires.

Plain water has a different dielectric constant than water containing DNA.

The microprocessor charges the capacitor by turning on an output bit

connected to one of the wires. The other wire connects to ground.

Then, the output bit is switched to an input bit and compared to a voltage

reference, while a high speed timer counts how long it takes the capacitor

to discharge.

You can get very high resolution using this technique -- as many bits as your

timer has (usually 16) or even more if you use software to extend the timer

range to 24 or 32 bits. But even 8 bits is overkill for the DNA quantification

application, where 10% accuracy seems to be the norm, and three significant

digits are hard to find in the literature.

Calibration can be done by comparing to a Nanodrop or similar device, or

by making a pure DNA solution, removing the water, and then diluting it to

specified concentrations.

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[John Griessen]

On 12/05/2011 12:18 PM, Simon Quellen Field wrote:
> Simply measure the capacitance of the sample.
>
> Measuring capacitance with a microcontroller is very simple.
> Two wires, or two traces on a PC board make the capacitor.
> The dielectric is the sample placed across the wires.
> Plain water has a different dielectric constant than water containing DNA.
> The microprocessor charges the capacitor by turning on an output bit
> connected to one of the wires. The other wire connects to ground.
>
> Then, the output bit is switched to an input bit and compared to a voltage
> reference, while a high speed timer counts how long it takes the capacitor
> to discharge.

Soil scientists use a meter with a steady high frequency output to
electrodes so they have the outside world in between them. The impedance
is related to the different dielectrics of sand, clay, water and water is
the dominant one, so such probes tell you about water content fairly well
without calibration. With cal for a certain soil makeup, even better.

So, instead of a time measure, they do a ratio of volts to a known impedance
reference and output that as an analog voltage usually.

It's the same principle.

Chemists use
Electrochemical impedance spectroscopy, (EIS), and potentiostats to study reactions.
impedance spectroscopy I'd never heard of -- that would be tuning through
many frequencies while measuring ratios like the soil moisture probes.

The potentiostat sounds like it clamps at a voltage, but they are really used to sweep
voltages, so it is akin to EIS above. I came across a bio use of a potentiostat to
ionically bond some methylene blue and measure that effect (an impedance to current flow
at 100Hertz sweep rate). The methylene blue was attached to some DNA binding molecule,
so it could detect a certain kind of DNA in a solution.

I'm going to figure out some good lab tools to help do these kinds of measurements.

Maybe a combination electroporator EIS and potentiostat controller! I'd probably put it
in different packages, with different cuvettes or sample wells, and the same controller
hardware and different code, but all open so if you wanted to hack, you could.
Maybe some would like the "Swiss knife" style with
electroporator/EIS/potentiostat/percent-moisture in one user interface.

An open virtual instrument GUI would be nice...

[PCR Tests]

Build it. That sounds great.

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[Robert O'Callahan]

Now this is a really interesting patent:
http://www.google.com/patents?id=j6rVAAAAEBAJ
qPCR using capacitance to quantify DNA

-Rob

[John Griessen]

On 12/05/2011 02:49 PM, Robert O'Callahan wrote:
> Now this is a really interesting patent:
> http://www.google.com/patents?id=j6rVAAAAEBAJ
> qPCR using capacitance to quantify DNA

That seems very similar to the cheapstat folks' method (fig 6.),

http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0023783

but they do it between PCR cycles in the combination they patented.

As long as you used individual instruments and probes you hooked up
in your own lab, the patent might not apply to the individual parts.
They ask for patent of a system and method though -- if method means
process patent, it could get in the way of research done this way.
They probably want to sell to researchers and not stop them...

[jlund256]

> > qPCR using capacitance to quantify DNA

This is using the *change* in capaitance to measure the change in DNA
concentration. Using absolute capacitance to measure concentration is
unlikely to work--DNA solutions are mixtures of DNA, nucleotides,
protein, salts, other bits of DNA, carbohydrates, and more. It is
rare that a DNA solution is pure enough that DNA will be the main
conductor. At least that's my guessimate, it would be great to see
data. If someone wants to experiment with the system and report back
that would be great.

Jim Lund

[Ashley Heath]

There is also an instrument, see http://www.sharplabs.com/biosensor.php.

[Nathan McCorkle]

Also these techniques (at least the patent and the PLoS linked ones)
require an oligo probe for hybridization, making either system less
appropriate for general DNA quantification.

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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[John Griessen]

On 12/06/2011 09:45 AM, Nathan McCorkle wrote:
> DNA solutions are mixtures of DNA, nucleotides,
>> protein, salts, other bits of DNA, carbohydrates, and more. It is
>> rare that a DNA solution is pure enough that DNA will be the main
>> conductor.

Sure, the approach Simon suggests is not an absolute measure, but it can
be a good relative one with a calibration from first principles.
And you'll need to sweep several frequencies, and measure DC conductivity
to weed out unknowns in a solution.

Suppose you have a water solution of mostly DNA, with low low DC conductivity --
low ions -- good for a first principles calibration. What would happen if you
dry out DNA like Simon suggested? Would it return to anything like it was
after drying out to something like 20% water -- a sticky blob level of dryness?

[John Griessen]

On 12/06/2011 09:42 AM, Ashley Heath wrote:
> There is also an instrument, see http://www.sharplabs.com/biosensor.php.

They say,
"Physically, impedance changes are caused by changes in the near-surface environment of the electrode and, thus, provide a direct
means of detecting target-probe binding reactions on the sensor."

so this is also a bit of DNA bound as a probe to an electrode -- not general impedance
measuring to imply concentration.

Simon was suggesting measurements without any special DNA laced electrodes.

[mad_casual]

My experience, I'd say it's a concerted effort (rather than rare) that
produces a desalted DNA solution, but you're right. Part of the beauty
of spectrophotometric methods is that the solution can be full of
other crap and still work. This explains the prevalence of fluorophore-
based array solutions and the paucity of electronic-based solutions. I
do wonder, though, if it would be possible to create a desalting or
mini-prep(ish) tube setup that tells you the relative yield or some
estimate of "% purification". I know ISEs are used in column
chromatography to similar effect.

[Simon Quellen Field]

The DNA is not a conductor in the capacitance probe.

It is the dielectric.

The probe tips can be insulated, and the trick still works.

The calibration is by comparing to samples done using similar techniques,

but with different DNA concentrations, as measured by a standard test instrument.

This means that the thing that differs between the tests will be the amount

of DNA, with other factors remaining the same, to within the 10% limits of

resolution required.

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[Nathan McCorkle]

I wonder if whether through capacitance, or RF resonance I could
detect growth in a DNA strand, 1 nucleotide at a time. It would be a
sure way to realize if a nucleotide addition occurred after a
synthesis step

This might apply to the latter idea:

Stepwise oscillatory circuits of a DNA molecule - Kunming Xu

Abstract: A DNA molecule is characterized by a stepwise oscillatory
circuit where every
base pair is a capacitor, every phosphate bridge is an inductance, and
every deoxyribose is
a charge router. The circuitry accounts for DNA conductivity through
both short and long
distances in good agreement with experimental evidence that has led to
the identification of
the so-called super-exchange and multiple-step hopping mechanisms.
However, in contrast
to the haphazard hopping and super-exchanging events, the circuitry is
a well-defined charge
transport mechanism reflecting the great reliability of the genetic
substance in delivering
electrons. Stepwise oscillatory charge transport through a nucleotide
sequence that directly
modulates the oscillation frequency may have significant biological implications.

http://mel.xmu.edu.cn/group/ocg/admin/uploadfile/2010122145530-440nOf.pdf

[Meow-Ludo]

If it could, you could probably sequence this way, as each base has a different charge.

BRB, patent. 

[Nathan McCorkle]

I really just care about realizing if the DNA has become longer by 1
base, and I'm talking about 1 base on a single molecule of DNA (not a
bunch like in a pot of PCR brew)

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[John Griessen]

On 08/22/2012 03:31 AM, Nathan McCorkle wrote:
> I really just care about realizing if the DNA has become longer by 1
> base, and I'm talking about 1 base on a single molecule of DNA

The big guns with patent protection are working that one as:
paraphrased -- 'DNA is forced electrochemically through a keyhole in a compound
that also acts as a sensor'. Without forcing things to happen in
a specific constrained location, the signal is too tiny I guess.

[Nathan McCorkle]

I specifically don't want to move the DNA... hmm...

Nucleotide Capacitance Calculation for DNA Sequencing
Jun-Qiang Lu and X.-G. Zhang
Biophys J. 2008 November 1; 95(9): L60–L62.
Published online 2008 August 15. doi: 10.1529/biophysj.108.140749
PMCID: PMC2567940
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567940/

"
Using a first-principles linear response theory, the capacitance of
the DNA nucleotides, adenine, cytosine, guanine, and thymine, are
calculated. The difference in the capacitance between the nucleotides
is studied with respect to conformational distortion. The result
suggests that although an alternate current capacitance measurement of
a single-stranded DNA chain threaded through a nanogap electrode may
not be sufficient to be used as a standalone method for rapid DNA
sequencing, the capacitance of the nucleotides should be taken into
consideration in any GHz-frequency electric measurements and may also
serve as an additional criterion for identifying the DNA sequence.
"

Maybe the author will respond to e-mail... I can't tell if quantifying
a single molecule of N bases works or not, since the paper is so
interested in sequencing

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[Heather]

Sorry to revive a very old thread - but I am wondering if there was any further development/experimenting with this capacitance method? I am looking for a DIY method to quantify DNA with minimal expensive equipment. Wondering if a capacitance meter would do the trick? Has anyone tried this and documented it anywhere? Or any other ideas on quick and dirty methods for quantifying or even just visualizing DNA without going through PCR and gel electrophoresis?

thanks!

Heather