ABCMdb

PMID: 15744517

Murphy D, Redmond G
Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide-nanoparticle conjugates.
Anal Bioanal Chem. 2005 Mar;381(6):1122-9. Epub 2005 Mar 3., [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Before using each oligonucleotide, a working stock solution was prepared from the sample received from the supplier Fig. 1 Probe and target oligonucleotide sequences relating to the four cystic fibrosis mutations investigated in this study, namely DF 508, M470D, R74W and R75Q mutations. Login to comment 81 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln The cystic fibrosis-related mutations that were selected for study are as follows: D F 508-a three base deletion of CTT at position 508 of the CFTR gene; M470V-an A to G SNP at position 1540 of the CFTR gene; R74W and R75Q-C to T and G to A SNPs at positions 352 and 356, respectively, of the CFTR gene. Login to comment 113 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln To explore the applicability and usefulness of oligonucleotide-nanoparticle conjugates for this purpose, the CF-related SNPs R74W (C to T) and R75Q (G to A) were selected for study. Login to comment 116 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Denaturation behavior was then investigated for complexes formed using either of four target strand sequence types-mutation-free, R74W-bearing, R75Q-bearing and a sequence bearing both mutations. Login to comment 121 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Concerning the melting behavior of the latter complexes, it is observed that dissociation of the R74W-bearing complex (red line) may be clearly distinguished from that of the R75Q-bearing complex (pink line). Login to comment 126 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Denaturation behavior of the hybridized nanoparticle aggregates was investigated for complexes formed using either mutation-free, R74W-bearing, or R75Q-bearing target strands of 50 and 72 base length. Login to comment 138 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln The second and third rows, each labeled ''DF 508`` and ''No mutation``, show Fig. 3a-c R74W and R75Q mutation detection. Login to comment 139 ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln a Thermal dissociation curves for three-strand complexes comprising two oligonucleotide-nanoparticle conjugates and a target oligonucleotide with the following sequence: (blue) mutation-free, (red) R74W mutation, (pink) R75Q mutation, and (brown) both R74W and R75Q mutations, respectively. Login to comment 141 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Also, thermal dissociation curves are shown for three-strand complexes comprising two oligonucleotide-nanoparticle conjugates and a target oligonucleotide with the following sequence: (blue) mutation-free, (red) R74W mutation, (pink) R75Q mutation, respectively, for b 50 base and c 72 base targets the denaturation behavior of nanoparticle aggregates formed using target strands of 27 and 24 base length, the former lacking the CTT deletion mutation and the latter bearing the mutation, respectively. Login to comment 144 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln This method was also applied to SNP mutation detection using the R74W and R75Q oligonucleotide sequences and aggregate hybridization conditions previously described for solution-based mutation detection experiments. Login to comment 145 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Denaturation behavior of hybridized nanoparticle aggregates was monitored for complexes formed using either of four target strand sequence types-mutation-free, R74W-bearing, R75Q-bearing and a sequence bearing both mutations. Login to comment 151 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Concerning the melting behavior of the aggregates formed using SNP-bearing target strands, it is observed that dissociation of the R74W-bearing complex (third row, 42 °C) may be clearly distinguished from that of the R75Q-bearing complex (fourth row, 33.5 °C) and that melting of the complex formed using the target sequence bearing both mutations may also be very clearly identified at the lowest melting temperature (fifth row, 28.5 °C). Login to comment 157 ABCC7 p.Arg74Trp ABCC7 p.Arg75Gln Using this approach, fully complementary sequences were successfully distinguished from mismatched sequences, with single base mismatch resolution, for DF 508, M470V, R74W and R75Q mutations. Login to comment