[Mutation scanning of short amplicons: optimization of DNA melting conditions].
High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.
I V
IV
Botezatu
K I
KI
Zhordania
A I
AI
Karseladze
A M
AM
Stroganova
B N
BN
Kondratova
V P
VP
Shelepov
M V
MV
Telkov
A V
AV
Likhtenshtein
D052160:Benzothiazoles; D003110:Colonic Neoplasms / Q000175:diagnosis / Q000235:genetics; D004247:DNA / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D003959:Diamines; D005188:False Negative Reactions; D005260:Female; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics; D016612:Paraffin Embedding; D012150:Polymorphism, Restriction Fragment Length; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D044366:Transition Temperature
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D005557:Formaldehyde; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras)