J Preiss--Seq Anal
preissj at CLVAX1.CL.MSU.EDU
Tue Oct 27 19:30:00 EST 1992
Obviously there are some serious misunderstandings concerning nucleic acid
denaturation. As was shown approximately 30 years ago, formaldehyde does not
affect the stability of duplex nucleic acid, (monitored as a change in Tm)
but, upon reacting with the free amino groups exposed during denaturation,
obstructs the renaturation process (Stollar and Grossman). This is different
than the effect of formamide, which affects the Tm of nucleic acid directly.
(Casey and Davidson) The reaction of an aldehyde with an amino group produces
a Schiff base adduct, which is very labile at pH greater than 7.0 (less than
the pH of most nucleic acid electrophoresis buffers). Presumably glyoxal
behaves comparable to formaldehyde in relation to the amino groups involved in
base pairing. Glyoxal produces an additional more stable lesion in a reaction
with guanine bases. (Broude and Budowsky) Thus the standard protocols have
denaturation of the RNA with mild temperature in the presence of the aldehyde
reagent of choice followed by electrophoresis through a buffer which will
either not hydrolyze the stable adduct (for glyoxal treated RNA) or will
provide an environment conducive to maintaining the Schiff base adduct by
driving the equilibrium toward adduct formation (for formaldehyde treated
RNA). The failiure to denature secondary structure will result in
electrophoretic problems (the sieving properties of equal mass objects depends
significantly upon the shape of the object) as will the failure to maintain
the denatured state (changing the physical condition of the RNA during
electrophoresis). The foregoing is pertinent to any single-stranded nucleic
acid (for example consider DNA sequencing gel protocols which utilize the
reaction of urea with exposed carbonyl to obstruct secondary structure of the
DNA fragments to be resolved).
The removal of both Schiff base adducts occurs at pH greater than 7.0.
The stable guanine adduct is effected by heating to 60C at pH 8.0 (Thomas)
after the nucleic acid is transferred to the membrane of choice. Failure to
remove these should interfere with renaturation of the probe with the
immobilized target nucleic acid (Stollar and Grossman). I do not know of
anyone who willingly admits to not removing the adducts either during or after
I have examined the amount of nucleic acid transferred from a
formaldehyde gel to S&S NYTRAN by fluid using a vacuum transfer device for 20X
SSC (classic transfer vehicle), 7.5 mM NaOH (modification of BIO-RAD), 7.5 mM
NaOH for 1/3 time followed by 0.1 X SSC, and electrotransfer. I find the
relative amount of nucleic acid transfered to the membrane from gels which had
the same amount of RNA loaded to be in the ratio of 1:3:5:50 for the above
methods. 50 is a minimal estimate.
Stollar and Grossman, (1962) JMB 4, 31-38.
Casey and Davidson, (1977) Nuc. Acids Res. 4, 1539.
Broude and Budowsky, (1971) BBA 254, 380-388.
Thomas, (1983) Methods in Enzymology 100, 255-266.
BIO-RAD - User manual for ZETAPROBE membrane.
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