Nucleic Acids Research, Vol 25, Issue 19 3783-3786, Copyright © 1997 by Oxford University Press
P Ordoukhanian and JS Taylor
The ability to study the structure-activity relationships of the cis- syn
thymine dimer, the major photoproduct of DNA, has been greatly aided by the
availability of a building block suitable for its sequence- specific
incorporation into oligonucleotides by standard automated DNA synthesis.
Unfortunately, its usefulness is compromised by the fact that it takes six
steps to synthesize in low overall yield and, as with all phosphoramidite
building blocks, has to be used in great excess over the support in
standard automated synthesis. To extend the usefulness of this building
block, we have directly coupled it to standard A, C, G and T long chain
alkylamine-linked controlled pore glass supports to yield a solid
phase-supported dimer. We then demonstrate that 13mers containing a
3'-terminal d(T[cis-syn]TN) group synthesized with this support at 0.2
micromol scale can be efficiently incorporated into longer oligonucleotides
by both primer extension with 3'-->5'exonuclease-deficient Klenow
fragment or T4 polymerase and dNTPs or by enzymatic ligation with T4 DNA
ligase to another oligonucleotide opposite a complementary template. The
site specificity and integrity of the cis-syn thymine dimer after both
primer extension and ligation was confirmed by cis-syn dimer-specific
cleavage with T4 denV endonuclease V. This general approach should be
applicable to the synthesis of many types of site-specific nucleic acid
modifications and would be of particular use for those for which the
required building blocks are expensive or difficult to make.
ARTICLES
Solid phase-supported thymine dimers for the construction of dimer- containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA
Department of Chemistry, Washington University, St Louis, MO 63130, USA.
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