Introduction: In recent times, interest in genetic modification called for the
development of important techniques in the analysis of modified oligonucleotide
sequences. Many DNA sequence detection methods are known to date, including the
use of DNA chips and real-time PCR methodology techniques. However, they all
require target amplification and/or labelling steps during analysis. The method
developed herein allows the qualitative and quantitative detection of DNA sequences
without the need for target amplification or labelling steps prior to the analysis, but is
based on the site specific DNA cleavage on solid-support.
Method: Akin to other currently employed techniques, our innovation makes use of
an immobilised DNA strand. However, we incorporate a photocleavable site X, in
addition to a marker tag M at the terminus of the strand. Together with the target
sequence, a double helix is formed and upon irradiation, X is cleaved and the marker
containing fragments remain on the support through base pairing with the
complementary target strand. In case of mismatches, located either in the upper or
lower section, the stability of the double strand is attenuated, and releasing the
fragment containing the marker tag M, which subsequently is detectable in solution
(Figure A).
Results: For the construction of the device we made use of the disulfide exchange
mechanism which lead to quantitative yield for the immobilisation of the used 3’ modified
oligonucleotides (Scheme A).
Furthermore, hybridisation of counter strands to SP-B showed good yields. After
heating to 85 °C and cooling for a minimum of 1.5 h with stirring, quantitative
hybridisation yields were obtained.
Irradiation of an X and M modified single strand SP-B affords quantitative yield of
cleavage produkt ON-B (Scheme A). Identical irradiation experiments using a duplex
with the complementary strand did not release the same amount of ON-B. The main
part of ON-B (~80%) remaining on the support could be released upon heat treatment.
This is the expected „counter-strand-effect“. Introduction of two mismatches into the
counter strand lead to a destabilisation of the helix and therefore deliberation of ON-B
indicating the mismatches. Incorporation of only one mismatch gives various results,
but it could be shown that at selected positions single mismatches (SNPs) can be
detected using our methodology. With the obtained “counter-strand-effect” the potential
of the herein presented method for qualitative sequence detection could be shown.
Furthermore, the effective strand cleavage on the solid support offers the opportunity
for quantitative sequence detection (e.g. for GMO evidence)
