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Custom DNA Microchip Nucleotide

A modified base for use in DNA microchips to promote more even hybridization conditions.
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DNA microchips are small wafers that contain thousands of short DNA probes, about 20 or so nucleotides in length. They all correspond to genes. By washing cell extract over the microchip, those genes being actively synthesized will anneal to their complimentary probe due to sequence specificity. They anneal to form a small double helix, and the presence of an annealed product can be measured by optical absorbance.

Recently in one of my genomics lectures, the prof said that they don't work as well as people often think, because of the difference in base pair binding. A-T pairs bind with 2 hydrogen bonds, while G-C pairs bind with 3, meaning a probe with large amounts of G-C content would hybridize more strongly than a largely A-T probe, because of the greater number of H-Bonds. Thus, it is hard to find a common temperature for the experiment if both of these kinds of probes are on one chip.

My suggestion here is to make modifications to Guanosine (G) and Cytosine (C) to produce new nucleotides. These are incorporated into the DNA probes in the place of G and C. The modifications result in only 2 H-bonds between modified G and cellular C, and vice versa. Alternatively, probe G and C could be made bulkier to produce steric interference while still retaining their 3 H-bonds with cellular C/G, effectively raising the energy state of hybridized C-G to that of A-T.

Making G-C binding energy comparable to A-T binding energy reduces restrictions placed on probe sequences.

The only problem here is that I don't have the organic chemical knowledge or mathematical tools to accurately compute what a change in one atom would do to the energy of hybridization. My first guess would be to replace N-5 in cytosine with Carbon, and the complimentary Nitrogen in Guanosine with Carbon as well.

O-Chemists...any ideas? (Mr angel chaser, I'm looking at you.)

Cuit_au_Four, Apr 12 2008

Base Pairing http://fig.cox.miam...ene/BasePairing.gif
Diagram showing the base pairs. [Cuit_au_Four, Apr 12 2008]

a liquid crystal chemical structure http://www.google.c...uid+crystal+formula
just tether the amino acid to this n apply charge; study if it affects affinity [beanangel, Apr 14 2008]

water with magnet plus lipid is an order opportunity http://www.otherpower.com/diamagh2o.html
gorgeous photo of water with magnet [beanangel, Apr 14 2008, last modified Feb 04 2012]

Nanogen http://www.nanogen....ologies/microarray/
Electronic control of hybridisation [MaxwellBuchanan, Apr 14 2008]


       Yo [Cuit]. An excellent idea, but alas extensively baked, or at least undergoing baking. People have tried modifying all the bases to make the annealing temperatures more uniform, with limited success. Other strategies have included modifying the buffer conditions (to favour A/T pairing and make it more similar in energy to G/C pairing), and also replacing some bases with 'universal' bases, leaving the other bases unaltered to confer specificity. Some of this work goes back to the late 80's/early 90's when "sequencing by hybridisation" was being tried, and there's been a resurgence of interest since the blossoming of array technologies. Similar issues arise in PCR primer design, and again modified bases have been tried.   

       As far as I know, success has been limited. Nowadays, it's more usual to put the effort into probe selection (since you normally have a wide choice of available sequences for any given target region) to try to achieve uniform Tm's.   

       Also, factors other than base composition can greatly affect hybridisation. Secondary structure and "base stacking" (interactions along the strand rather than between strands) can have considerable impacts.   

       An excellent idea, nevertheless.
MaxwellBuchanan, Apr 12 2008

       (feels desperately inadequate to address this idea) Um.... I think if we use pulleys and add in a few spacer washers it should work.
RayfordSteele, Apr 12 2008

       as ignorant as this will sound has anyone tried either liquid crystals decorated with nucleotides that can have their electron density graph adjusted with a current like liquid crystals   

       basically with liquid crystals the molecular fronds plump or cluster or compress with charge application; with repeated testing this effect might provide either the electron density graph modification to equalize the (CG) (AT) difference or actually slightly sequester, kind of hide reminiscent of protein, the (CG) (AT)ness to equality   

       another possibility is putting a teeny electric circuit with each dot of the array to see if electrochemistry has the desired effect   

       then, just to be wild you could try magnetism yep view [link] where diamagnetic water migrates to the side of a glass with a giant magnet; the hydration pattern around (CG)(AT) is certainly modifiable with magnetism but you gotta view the [link]   

       a blend of diamagnetic water with another solvent could create nonthermal magnetically wiggleable amino acid reaction points   

       rather than run the reaction at a temperature to use thermal motion to produces links you'd vibrate a diamagnetic plus paramagnetic solvent with em
beanangel, Apr 14 2008

       Oddly enough, that has been tried. There's a company called Nanogen that, maybe 8 or 10 years ago, developed a chip consisting of a set of independently-addressable electrodes. An applied voltage could be used to attract an oligo to each pad, where it could be attached chemically to act as the target (thereby potentially simplifying the step of making the chip).   

       More significantly, the applied voltage could then also be used to attract the probe to specific targets on the array, or to drive the probe away, thereby providing a sort of electronic control over hybridisation stringency.   

       I don't think they have as high a feature density as conventional arrays, but the voltage control is a neat trick. See link.
MaxwellBuchanan, Apr 14 2008

       I recently read a patent that used 0 to 100 micronewtons of force to do PCR cycling rather than warm cool cycles. I wonder if that would work with a blend of diamagnetic water with another solvent could create nonthermal magnetically wiggleable amino acid reaction points to create magnetic PCR   

       It could go with genechips if the genechips had their ligands on lipid c-c-c-c-c-c-c-c-c-c-c-c-c-c thingies giving the gene chip the ability to be electronically (magnetically) contrast enhanceable
beanangel, Feb 04 2012

       What was the patent? And how was the force applied?
MaxwellBuchanan, Feb 04 2012


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