User:Justinhlo

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(Mismatches)
(Mismatches)
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</pre>
</pre>
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The original sequence has 8 G-C pairs, or 42%.  I think this is pretty normal, and it may be redundant to bother with different G-C contents, since it's pretty well-established that the more G-C, the higher the melting temperature (presumably due to hydrogen bonding effects?).  One thing I do wonder about is the relationship of G-C content to ionic strength sensitivity of the T<sub>m</sub> values, since the triple H-bond format could interact differently with Na/K as compared to the 2-H-bond format.  Anyway, that isn't that important, probably.
+
The original sequence has 8 G-C pairs, or 42%, which is in the middle of what seems to be the normal 20-60% range (for proks, anyway .. http://insilico.ehu.es/oligoweb/index2.php?m=all).  I think this is a good representative sequence, and it may be redundant to bother with different G-C contents, since it's pretty well-established that the more G-C, the higher the melting temperature (presumably due to hydrogen bonding effects?).  One thing I do wonder about is the relationship of G-C content to ionic strength sensitivity of the T<sub>m</sub> values, since the triple H-bond format could interact differently with Na/K as compared to the 2-H-bond format.  Anyway, that isn't that important, probably.
The total mismatch case used the sequence:
The total mismatch case used the sequence:
Line 53: Line 53:
19bp total mismatch
19bp total mismatch
5'- ATCAA GCAGC CATGC AAA -3'
5'- ATCAA GCAGC CATGC AAA -3'
-
3'- TATTC TGTTC CTGGT TTCC -5'
+
3'- TATTC TGTTC CTGGT TTCC -5'
 +
      ^^^ ^^ ^^ ^ ^^^  ^^
</pre>
</pre>
I do not know if the top line is a typo or not .. it doesn't actually have 19 bp ...
I do not know if the top line is a typo or not .. it doesn't actually have 19 bp ...
 +
 +
This is not really a "total mismatch," but even if it hybridizes, (at the ends), it will probably always show up as ssDNA due to the behavior of the intercalating dye.
===Length===
===Length===

Revision as of 14:11, 8 January 2007

Contents

DNA Melting Project: IAP 2007

Ionic Strength

Using either NaCl or KCl (is there an advantage to one over the other? Does Na+ or K+ interact more with O-?). Original 19-bp sequence is fine ..

Goal: show how the set-up can be used to investigate the significant effects of ionic particle concentrations on DNA melting parameters.

Control and Experimental Groups:
0 mM (control)
1 mM
10 mM (original module condition)
150 mM (physiological conditions, see
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2703503&dopt=Abstract)
1000 mM (SantaLucia paper’s conditions – I’m curious to see if we get similar results).

These conditions form an approximately logarithmic distribution between 0 M and 1 M. This is justified because the projected dependence of ΔS and ΔH on ionic concentration is also logarithmic. The 150 mM may be replaced by 100 mM if it is deemed more desirable.

It may be worth looking into the 50 mM concentration, because this is what PCRs are run at (50 mM KCl, specifically), and it is also what many of the models have actually been designed for.

Mismatches

C, T are pyrimidines (small)
A, G are purines (large)

The original 19-bp sequences that we tested were:

19bp perfect match:
5'- ATCAA GCAGC CATGC AAAT -3'
3'- TAGTT CGTCG GTACG TTTA -5'

19bp single-base mismatch (SNP) [T-C mismatch]:
5'- ATCAA GCATC CATGC AAAT -3'
3'- TAGTT CGTCG GTACG TTTA -5'
             ^

The mismatch is a pyrimidine-pyrimidine type. I would be curious to compare that to an SNP of purine-purine type would increase the differences, so for instance:

19bp single-base mismatch (SNP) [A-G mismatch]:
5'- ATCAA GCAGC CATGC AAAT -3'
3'- TAGTT CGTAG GTACG TTTA -5'
             ^

The original sequence has 8 G-C pairs, or 42%, which is in the middle of what seems to be the normal 20-60% range (for proks, anyway .. http://insilico.ehu.es/oligoweb/index2.php?m=all). I think this is a good representative sequence, and it may be redundant to bother with different G-C contents, since it's pretty well-established that the more G-C, the higher the melting temperature (presumably due to hydrogen bonding effects?). One thing I do wonder about is the relationship of G-C content to ionic strength sensitivity of the Tm values, since the triple H-bond format could interact differently with Na/K as compared to the 2-H-bond format. Anyway, that isn't that important, probably.

The total mismatch case used the sequence:

19bp total mismatch
5'- ATCAA GCAGC CATGC AAA -3'
3'- TATTC TGTTC CTGGT TTCC -5'
      ^^^ ^^ ^^ ^ ^^^   ^^

I do not know if the top line is a typo or not .. it doesn't actually have 19 bp ...

This is not really a "total mismatch," but even if it hybridizes, (at the ends), it will probably always show up as ssDNA due to the behavior of the intercalating dye.

Length

[...]

Potential Models for DNA modeling

This interesting paper actually lists a good number of methods (including the one we used in the module). http://bioinformatics.oxfordjournals.org/cgi/content/full/21/6/711. However, while it compares the methods thoroughly, it does not run the empirical experiments in order to see which one is actually right.

Here are a few models worth investigating: The very basic equation used for very short sequences: Tm= (wA+xT) * 2 + (yG+zC) * 4.

The standard G-C content equation: Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC).

The modified G-C content equation, with more length-dependent consideration: Tm = 100.5 + 41*(yG+zC-836.4)/(wA+xT+yG+zC).

The NN model: It seems that the last one here is probably the only one that actually tries to include entropic and enthalpic conditions. That means we have limited options for the fitting of the actual curve.

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