From: David J. Scott <djs17@york.ac.uk>
  To  : rasmb@bbri.harvard.edu
  Date: Mon, 13 Jul 1998 13:03:11 PDT

DNA vbar summary

Hello RAMB'ers.
 I have collated some of the replys for my question on calculating vbar of DNA. Unlike (most) proteins, it appears that it 
depends crucially on buffer type, pH and salt concentration, complicating the problem somewhat.  Some workers simply 
take the average value (c.a. 0.55), while others performing more elegant theoretical calculations. Experimentally, there is 
of course the classic method of direct measurement of vbar by comparison of equilibrium distributions in H2O and D2O, 
which we will probably be attempting first. 

Thanks to all for the contributions :-)

Dave Scott.
 




Olwyn Byron wrote:

The vbar of DNA can be calculated from the constituent bases as for amino
acids but it is very sensitive to salt and pH. Here are some data vbars
that we measured some time ago. I sent them to Tom Laue last November and
they might be of some use to you now.

The data are for (single stranded) polyX at ambient temp (about 20C).

oligo  in Tris   in phosphate   in HEPES   in TEA
A       0.5219    0.4765         0.5362     0.5287
C       0.5666    0.5286         0.5841     0.5613
G       0.6193    0.5513         0.6102     0.5548
T       0.5227    0.4527         0.5522     0.5498
U       0.4061    0.5431         0.4978     0.5443


Best wishes,

Olwyn.

**************************************************************
Frank Hays wrote:

we use john philos SEDNTERP program to calculate vbars from aa
compositions.  it is readily available via the RASMB ftp site...
hope this helps some
frank

********************************************************************************
Franklin A Hays
Analytical Ultracentrifuge Technician
Department of Biochemistry and Molecular Biology
246 NRC
Oklahoma State University
Stillwater, OK 74078
haysf@biochem.okstate.edu
405.744.9322/4138
********************************************************************************


Jack Lebowitz wrote:

The partial specific volume (1/buoyant density) varies with the G-C
content of the DNA. In the past buoyant density measurements were
performed in CsCl in order to determine the G-C content for large DNAs .
In 1980 we (Woodward, R.S. and Lebowitz, J.) published a paper in
Journal of Biochemical and Biophysical Methods 2, 307-309 entitled a
Revised Equation Relating DNA Buoyant Density to Guanine Plus Cytosine
Content. I am not sure that the above journal survived. The equation to
calculate the buoyant density is 
Theta (buoyant density)=0.0988(fraction G-C content)+1.6541
We have used the above equation in recent DNA protein binding studies
with success. However, since a small DNA may not average out hydration
compared to a large DNA it is important to determine v bar in
sedimentation equilibrium experiment since you know the precise
molecular weight of DNA. 

Jack Lebowitz
 
Department of Microbiology
520 CHSB
University of Alabama at Birmingham
Birmingham AL 35294-2041
205 934-9475
FAX 205 975-4621

Forwarded Email from Tom Laue:::

>Return-Path: <chaires@fiona.umsmed.edu>
>Date: Wed, 12 Nov 1997 10:48:40 -0600
>From: Chaires <chaires@fiona.umsmed.edu>
>Subject: DNA partial specific volumes...
>X-Sender: chaires@fiona.umsmed.edu (Unverified)
>To: Tom.Laue@unh.edu
>
>
>Jack passed on you email about partial specific volumes of
>DNA.  For duplex DNA, some are listed in NaCl and CsCl on
>p.223 om Physical Chemistry of Nucleic Acids by Bloomfield
>Crothers & Tinoco (Harper & Row, NY, 1974).  Those data
>are taken from Cohen & Eisenberg (1968) Biopolymers 6:1077.
>
>I doubt very much if you will be able to find any numbers at
>all for single and triple stranded DNAs...
>
>Cheers,
>
>BRAD
>Dr. Jonathan (Brad) Chaires
>Department of Biochemistry
>University of Mississippi Medical Center
>2500 N. State St.
>Jackson, MS 39216-4505
>
>Telephone: (601) 984-1523
>FAX: (601) 984-1501
>Email: chaires@fiona.umsmed.edu
>

Tom Laue wrote:

Hi David,
Ahhh, the vbar of DNA. I'd wax poetic about it, but this problem is a ($^$@
to address. The problem stems from the fact that DNA is so highly charged.
This results in a vbar that is highly dependent on salt type and salt
concentration. There is also the matter of composition. All of this has
been studied (mostly in the 60s), but I can't put my hands on the papers.
Your best bet (short of measuring the vbar) is to get a hold of the papers
(look in the book by Crothers, Tinoco and Sauer- they have a lot of good
physical information about DNA in it, and a good reference list) and make
an educated guess based on your solvent type and the DNA composition. Also,
it isn't too hard to estimate vbar from equilibrium studies done in H20 and
D20 (even  better is to use O18-based D20). Since the molecular weight
doesn't change (hopefully!), the difference in the equilibrium
distributions comes from the buoyancy. Adjusting for the D/H mass exchange
(~5%, if memory serves me), the vbar can be extracted. This doesn't require
much material, and works well if your system is well behaved and you have
good lab hands.

*******************************
* Dr. David Scott		*
* Dept Biology		*
* University of York		*
* YORK			*
* YO1 5DD		*
*			*
* United Kingdom		*
*			*
* phone  +44 1904 432868	*
* fax       +44 1904 432860	*
*******************************