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Old 10-06-2009, 08:24 AM   #1
pmiguel
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Post What is at the ends of sonicated DNA?

Given the poor molar yields of typical next gen library construction methods, I have often wondered whether end-repair after shearing was an issue. There are a number of places along the phospho-deoxyribose backbone of a DNA strand where a shearing method might break the strand. Basically you have a repeating chain of (beginning at the 3' carbon and moving 5'):

C-C-C-O-P-O-C-C-C-O-P-O- etc.

So you could have C-C, C-O or P-O bonds "rupturing" to break the chain. I don't think any of the C-C breaks would be repairable by the typical end-polishing mixture of T4-DNA polymerase + T4-polynucleotide kinase. How about the others?

P-O rupture would likely result in one phosphoryl end and one hydroxyl end via the equivalent of hydrolysis. If it happens to be a 5' phosphate and a 3' hydroxyl, then no repair at all is needed--you have ends ligatable by T4-DNA ligase. If it is a 5' hydroxyl and a 3' phosphate then T4-polynucleotide kinase should kinase the 5' end and phosphatase the 3' end. The resulting ends, again, are substrates for T4-DNA ligase.

C-O rupture, as long as it leads to a hydrolysis-like result, would yield a similar prognosis. However, (see below) the result may not result in the restoration of the hydroxyl group via "solvolysis".

What type of ends do various shearing methods leave?

This question was addressed a paper that not only predates DNA sequencing of any kind, but also usage of agarose gel eletrophoresis to size DNA molecules:

Oliver C. Richards and P. D. Boyer, Chemical Mechanism of Sonic, Acid, Alkaline and Enzymic Degradation of DNA, J. Mol. Biol. (1965) 11, 327-340.

The take home was that sonication produced approximately 90% C-O bond rupture and 10% P-O bond rupture and no detectable C-C bond rupture, nor detectable removal of nitrogenous bases. The DNA sheared was phage T2 genomic. The authors give a MW of 1.6x10^8 daltons, or approximately 185 kbp, using 650 daltons as the MW of a basepair. They shear the DNA to an average of 492 +/- 92 bp in the first experiment and 492 +/- 15 bp in the second experiment. Here is there description of the sonication:

Quote:
Sonic irradiation of DNA was performed with a Branson 20 kc/sec probe-type sonic oscillator on solutions in SSC/10 at a concentration of 0.2 to 0.5 mg DNA/ml . The container with the DNA solution was immersed in an ice-water bath and sonic treatment was usually performed for periods of from 2 to 15 min at peak power of the probe in an air atmosphere or in a nitrogen atmosphere (after prior bubbling with nitrogen for a period of 5 min). Batches of 50 ml. in a 100-ml. beaker, or batches of 7 ml. in the thimble attachment for the Branson sonic oscillator were used.
The analytic methods used in the paper seem a little arcane to the modern reader. All molecular weight determinations were done via sedimentation velocity. Sites of bond rupture were determined by amount of incorporation of 18O oxygen isotopes into terminal phosphates during shearing as assayed by mass spec on inorganic phosphate released by alkaline phosphatase. But once you get past that, the paper is approachable enough.

The authors evidence against C-C bond rupture was that this type of break would not result in a terminal phosphate that could be released by alkaline phosphatase. But alkaline phosphatase did release phosphate in molar quantities equal to the number of fragment ends after shearing. Further, P-O bond rupture would result in 18O capture into terminal phosphates via "solvolysis". C-O bond ruptures, whether they proceed through hydrolysis or not, would not result in 18O being incorporated into the terminal phosphate. Two trials with sonication resulted in (8%, 12%, respectively for experiments 1 and 2) of the cleavage occurring at a P-O bond.

The figures for acid, base and DNase hydrolysis of DNA were 20%, 33% and 127%, respectively.

Should all sonication-sheared ends be T4-DNA ligase ligatable after T4-poly/T4-PNK end polishing? Based on the evidence presented by the authors, the majority of sonication breaks would be produced at C-O bonds. The author write:

Quote:
If, as seems likely, sonication breaks covalent bonds in the polynucleotide chain, C-O bond rupture must be predominant. Probably this occurs by solvolysis, with the introduction of water into the alcoholic group formed.
However, later in the discussion they continue:

Quote:
C-O cleavage may occur, however, without the uptake of medium oxygen by an elimination process.
They then consider and discard the possibility of a beta-elimination because it would result in nitrogenous base release--which they did not detect. They continue:

Quote:
Elimination not dependent upon a free carbonyl group remains possible, however. In this regard, Horwitz, Chua, Klundt, DaRooge & Noel (1964) have reported a base-catalysed elimination reaction of the pyrimidine nucleoside, 3'-O-mesyl-5' -O-trityl-2'-deoxyuridine, which results in the introduction of a 2',3'-double bond in the carbohydrate moiety. The uracilyloxy group behaves as the leaving group with the intermediate formation of bridge oxygen between the 3' position of the carbohydrate and the 2-position of uracil.
If I am interpreting this correctly, you could end up with a double-bond in the ribose backbone and no 3' hydroxyl. That, I would hazard, would not be ligatable by T4-ligase. Were it to form the terminus of a 3' overhang, it is conceivable that T4-polymerase's 3'-5' ssDNA exonuclease activity would remove it. But my guess is that it would not.

--
Phillip

Last edited by pmiguel; 02-15-2010 at 04:31 AM. Reason: Fix some typos
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Old 10-08-2009, 01:12 AM   #2
polivares
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Thanks !!! Very interesting, I'll will chew it around and discuss it with chemists. I'll report if there is something to share.
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Old 03-22-2010, 11:31 PM   #3
lvxiaobao
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Thanks a lot! Though i can't understand it exactly i think it's a really good post. By the way, can somebody explan the mechanism of t4 dna ligase? Thanks!
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Old 10-19-2011, 09:44 AM   #4
pmiguel
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Okay whippersnappers, more along the same lines for you in this paper:

END GROUP LABELLING OF RNA AND DOUBLE STRANDED DNA BY PHOSPHATE EXCHANGE CATALYZED BY BACTERIOPHAGE T4 INDUCED POLYNDCLEOTIDE KINASE; George Chaconas, Johan H. van de Sande and Robert B. Church; BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1975 Vol. 66, No. 3, pp 962-969

Check this out:

Quote:
SUMMARY : End group labelling of sheared double-stranded DNA, and tRNA has been effected without prior dephosphorylation, utilizing the reversal of T4 polynucleotide kinase activity. Incubation of DNA with polynucleotide kinase in the presence and absence of a phosphate acceptor (ADP) allowed the determination of the relative ratio of 5' hydroxyl and 5' phosphoryl terminii in the polynucleotide. This method of analysis has demonstrated a high preference in the formation of 5' vs 3' phosphomonoesters during high pressure shearing of double stranded DNA.
And, to hammer home the point:


Quote:
The great preference for the formation of 5' phosphoryl-terminated polynucleotides during high pressure shearing is similar to that observed for DNA fragmented by sonic irradiation (26, 27). This preference is expected due to the greater stability of the phosphate esters of primary versus secondary alcohols.

[...]

26. Richards, O.C. and Boyer, P.D. (1965) J. Mol. Biol. 11, 327.
27. Richardson, C.C. (1966) J. Mol. BioZ. 15, 49.
Previously I found the reported success rate for ligating untreated hydrosheared DNA inexplicable. But apparently the phosphate esters of primary alcohols are more stable than those of secondary alcohols.

Also, if you ever felt like the wind was behind your back during a library construction -- it actually was!

--
Phillip

Last edited by pmiguel; 10-19-2011 at 09:50 AM.
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Old 10-25-2011, 08:06 AM   #5
whw
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Great post. Any idea about DNA breakdown in vivo? Presumably DNAses make the chemistry a little different...
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