"high stringency" washes -NOT?
Kristian H. Jensen
mantar at biobase.dk
Sat May 10 05:07:18 EST 1997
Peter Nilsson (Peter.Nilsson at klinkemi.umu.se) wrote:
: Hello Jim.
: I can only add a biochemical explanation on the effect of ionic strength ([Na]) on the stability of the double helix: The negative charges on
: opposite strands in the backbone, the phosphate-groups, destabilize the helix by repulsing the the strands in the double helix from each other.
: This intrinsic instability is balanced by positivly charged ions (Na+) wich shields the negative charges on the backbone. With very high ionic
: strength one get not only shielding but also a ionic chemical bound between Na+ and P-.
: Also high ionic strength increases the hydrofobic interactions between the bases "inside" the helix, the base stacking, which also stabilize
: the interactions between the oppossite strands. Therefore, these two effects acting together gives a stabilization (higher Tm) with increased
: ionic strength. Decreasing the [Na] means higher stringency.
: Regards, peter.
: Peter Nilsson, Molecular Biologist
: Clinical Chemistry Department,
: Umeå University Hospital,
: S-901 85 Umeå,
Hi all you cyber dna netter!
Exactly, there are 3 major contributions to DNA hybridisation;
backbone repulsion; base stacing and hydrogen bonding between bases.
The first two are responsible for the increase in Tm with salt, as
repulsion is shielded and hydrophobic base stacking is enhanced.
Base hydrogen bondig only becomes "important" at low ionic strength,
hence the low salt high stringency Washes.
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