'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups.
We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.
Ohkubo
Akihiro
A
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Kasuya
Rintaro
R
Sakamoto
Kazushi
K
Miyata
Kenichi
K
Taguchi
Haruhiko
H
Nagasawa
Hiroshi
H
Tsukahara
Toshifumi
T
Watanobe
Takuma
T
Maki
Yoshiyuki
Y
Seio
Kohji
K
Sekine
Mitsuo
M
D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D005898:Glass / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide
C528645:6N-acetyl-8-aza-7-deazaadenine; D015342:DNA Probes; D000225:Adenine