Oligonucleotides containing a bromo or iodo group are prepared conventionally with the exception that deprotection is carried out in ammonium hydroxide at room temperature for 24 hours. Under these conditions, degradation of the halogen group was less than 2%.
Frequently Asked Technical Question
QUESTION: What methods are available for crosslinking DNA with DNA, RNA, proteins?
RESPONSE:The most widely used method seems to be using Br-dU and, more recently, I-dU (1). The substitution of photoreactive Br and I for the 5-Me group of thymidine is attractive since their radii are similar to that of the methyl group. These modifications do not significantly affect the binding of oligonucleotides with proteins. Br-dU is irradiated at 308nm and crosslinking is typically not greater than 40%. Crosslinking of I-dU at 308nm is higher but is optimal at 325nm. Laser excitation is preferred.
After the synthesis of oligonucleotides containing Br-dU or I-dU, care must be taken to avoid loss of the halogens. Even though both modifications are quite stable to ammonium hydroxide, it is sensible to play safe and carry out the deprotection at room temperature for 24 hours. Even better, use the UltraMild monomers and deprotect with potassium carbonate in methanol at room temeperature. The oligonucleotide products should be protected from light - plastic tubes and amber vials are safe. Gel electrophoresis should be carried out protected from light.
REFERENCE(S):
(1) M.C. Willis, B.J. Hicke, O.C. Uhlenbeck, T.R. Cech and T.H. Koch, Science, 1993, 262, 1255.
QUESTION: What are the extinction coefficients for propynyl-dC and dU? Also Halogenated dC and dU derivatives? Others?
RESPONSE:See: Table of Extinction Coefficients
REFERENCE(S):
B. Froehler, Gilead
M. Powell
R. Somers
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