Development of amplification-based technologies for enrichment of nucleic acids with difficult sequences or low-abundance point mutations

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http://urn.fi/URN:ISBN:978-951-51-0579-0
Julkaisun nimi: Development of amplification-based technologies for enrichment of nucleic acids with difficult sequences or low-abundance point mutations
Tekijä: Ho Huu, Tho
Muu tekijä: Helsingin yliopisto, lääketieteellinen tiedekunta, kliinisteoreettinen laitos
Opinnäytteen taso: Väitöskirja (artikkeli)
Tiivistelmä: The aim of this study was to utilize molecular biology knowledge to develop novel amplification-based technologies, which enable enrichment of challenging nucleic acid targets to a level sufficient for detection, including those with extremely long, GC-rich and/or repetitive sequences and low-abundance single nucleotide RNA variants in the excess of alternative variants. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to generate a novel amplification technology (HPE-PCR), which temporarily destabilize secondary structures, in order to enhance DNA polymerase extension over GC-rich sequences. Different GC rich target sequences in the human genome, extremely long Fragile X GGC repeats and Myotonic Dystrophy type 1 CTG repeats have been used as models to develop and validate this novel technology. In order to detect a low-abundance RNA variant, we devised a novel technology using a competitive Extendable Blocking Probe (ExBP) in the reverse transcription reaction for allele-specific priming with superior selectivity. In ExBP-reverse transcription, the mismatch priming site on the alternative variant is blocked by a perfectly matched ExBP. This initiates formation of a stable cDNA-RNA hybrid that completely blocks false cross-priming by the target specific primer. In experimental models, the ExBP-based reverse transcription assay allowed for detection of multiple mutation types on different genes in at least 1000-fold excess of wildtype RNA and detection was linear over a 4 log dynamic range. This technique not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression. In conclusion, we have established HPE-PCR and ExBP-RT techniques to enable enrichment of different nucleic acid templates that are currently challenging to detect by PCR, such as long, GC rich and/or repetitive sequences as well as low-abundance point mutations in a vast excess of wildtype alleles. These techniques expand the capacities of current PCR technology; provide versatile and convenient research tools and open many new possibilities for its applications in molecular diagnostics.Yleistajuinen tiivistelmä ei saatavilla toistaiseksi
URI: URN:ISBN:978-951-51-0579-0
http://hdl.handle.net/10138/153041
Päiväys: 2015-02-13
Avainsanat: medicine
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