Table 1. Results from Primer Extension with 12 and 16
quantity of
sequence selectivitya
primer
RNA (12)
RNA (12)
amino-DNA (16)
template (fmol)
+C/+T/+A/+G
100
500
500
>3:1:1:1b
76:13:7:4b
>99:1:1:1c
a Product distribution for extended primer. b After 27 h. c After 4 h.
suitable for direct genotyping of individual nucleotides mass
spectrometrically in RNA transcripts. An RNA transcript
with several hundred nucleotides was shown not to interfere
with MALDI-TOF MS. Our method requires only 3 strands
and has high sensitivity, which may be improved further by
miniaturization. The methodology may become useful for
genotyping tumors or viruses24 mass spectrometrically,25
particularly if RNA used for quantitative expression analysis
or identification of viruses via microarrays26 is simulta-
neously genotyped, providing key information on geno- and
haplotypes. Approximately 80% of all known virus are
retroviruses, such as HIV, Ebola virus, influenza virus,
including the H5N1 strain, arbovirus-B, and the hepatitis
viruses. Cancer diagnosis will benefit from expression
analysis of oncogenes efficiently combined with SNPs
detection.27
Figure 2. MALDI mass spectrum of surface-bound RNA primer
12 extended to 15, in an enzyme-free reaction templated by RNA
transcript 13 on a gold slide with 10; 500 fmol of template and
800 fmol primer were hybridized to the surface.
DNA-primer 16 with a 3′-terminal 3′-amino-2′,3′-dideoxy-
adenosine residue and the activated ribonucleotides occurred
faster, with full primer conversion in good base fidelity in
<4 h.
In conclusion, we report gold surfaces coated with self-
assembled monolayers displaying 3′-linked oligonucleotides
Acknowledgment. The authors thank Dr. E. Kervio (U.
Karlsruhe) for helpful discussions. This work was supported
by DFG (grant No. 1063/1-4 and FOR 434).
Supporting Information Available: Syntheses, template
sequence, protocols for gold substrate and its use, and NMR
and MS spectra. This material is available free of charge
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