Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.
Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA-RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy 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.
Minerva Foundation Institute for Medical Research, Helsinki, 00290, Finland htho@mappi.helsinki.fi.
Tho H
TH
Ho
Minerva Foundation Institute for Medical Research, Helsinki, 00290, Finland.
Kien X
KX
Dang
Haartman Institute, Department of Clinical Chemistry, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, Helsinki, FI-00029 HUS, Finland.
Susanna
S
Lintula
Haartman Institute, Department of Clinical Chemistry, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, Helsinki, FI-00029 HUS, Finland Helsinki University Central Hospital, HUSLAB, Helsinki, 00029 HUS, Finland.
Kristina
K
Hotakainen
Minerva Foundation Institute for Medical Research, Helsinki, 00290, Finland.
Lin
L
Feng
Minerva Foundation Institute for Medical Research, Helsinki, 00290, Finland.
Vesa M
VM
Olkkonen
Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, FI-00014, Finland.
Emmy W
EW
Verschuren
TJT Technologies Oy, Helsinki, 02360 Espoo, Finland.
Tuomas
T
Tenkanen
Department of Surgery, Helsinki University Central Hospital, Helsinki, 00029 HUS, Finland Research Program Unit, Translational Cancer Biology, University of Helsinki, Helsinki, FI-00014, Finland Haartman Institute, Department of Pathology, University of Helsinki, Helsinki, FI-00014, Finland.
Caj
C
Haglund
Children's Hospital, University of Helsinki, Helsinki, FI-00014, Finland.
Kaija-Leena
KL
Kolho
Haartman Institute, Department of Clinical Chemistry, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, Helsinki, FI-00029 HUS, Finland.
Ulf-Hakan
UH
Stenman
Minerva Foundation Institute for Medical Research, Helsinki, 00290, Finland Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, FI-00014, Finland Department of Women's and Children's Health, Karolinska Institutet, Stockholm, SE-17176, Sweden jakob.stenman@fimm.fi.
Jakob
J
Stenman
D000483:Alleles; D003062:Codon; D006801:Humans; D009154:Mutation; D015341:Nucleic Acid Probes; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D018631:ras Proteins / Q000235:genetics
D003062:Codon; C117307:KRAS protein, human; D015341:Nucleic Acid Probes; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins