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Chemically Modified DNA Probes That Significantly Improve Mismatch Discrimination
Author(s) -
Owczarzy Richard,
You Yong,
Moreira Bernardo G.,
Behlke Mark A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a946-b
Subject(s) - nucleotide , dna , base pair , guanine , pyrimidine , nucleic acid , chemistry , locked nucleic acid , context (archaeology) , melting temperature , oligonucleotide , crystallography , biology , stereochemistry , biochemistry , gene , materials science , paleontology , composite material
Recognition of mismatches in DNA or RNA duplexes is important in a variety of molecular biology techniques, e.g., SNP analysis, real‐time PCR. Mismatch discrimination is evaluated from the difference of melting temperatures ( T m ) between matched and single base mismatched duplexes. We examined these T m differences for various chimeric probes where some nucleotides were modified with locked nucleic acids residues (LNA). Our goal was to develop general guidelines for design of probes that would improve mismatch discrimination relative to native DNA probes. Melting temperatures and thermodynamic stabilities were determined from UV melting experiments. Mismatch discrimination was found to be similar in both Na + and Mg 2+ buffers. Triplet of LNA modifications with the central base of the triplet at the mismatch site gives the highest difference of melting temperatures between perfectly matched and mismatched LNA‐DNA duplexes. Alternatively, LNA modifications can be introduced at a purine nucleotide of the mismatch site and this improves mismatch discrimination. If a pyrimidine base is located at the mismatch site, the nearest‐neighbor nucleotides flanking the pyrimidine base should be modified. Exception to these rules is the G·T mismatch where any LNA modifications of the guanine nucleotide or its nearest‐neighbor bases must be avoided because LNA modifications decrease mismatch discrimination in this case. The results were found to be general and independent of sequence context.