Method for testing a mutant gene through real time polymerase chain reaction using inhibition of 5'-flap endonuclease activity
a technology of endonuclease activity and which is applied in the field of real-time polymerase chain reaction for detecting dna mutations, can solve the problems of difficult maintenance of stable annealing state, low tm value, and not always showing expected mutation detection ability of melting curves, etc., and achieve excellent sensitivity and specificity.
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example 1
ion of Amplification Curve of RT-PCR Reaction Using Double-Stranded Primer (DSP) System
[0076]This example is a confirmation test of amplification curve of RT-PCR using DSP / primer forming double strand through hybridization with the position from fourth base of 5′-end of a forward primer. TAMRA as a quencher and FAM as a reporter were linked to the 5′-end of the forward primer and 3′-end of a probe, respectively.
*Primers and probe for testForward primer:5′-TAMRA-gccgcgctggatgaactgatac-3′Reverse primer:5′-cggcctgaacagtgagcgaag-3′Probe:5′-ccggtatcagttcatccagcgc-FAM-3′
[0077]* RT-PCR conditions:
[0078]95° C., 5 min. (1 time)
[0079]95° C., 15 sec / 60° C., 40 sec / 72° C., 30 sec. (35 cycles)
[0080]* Composition for RT-PCR
[0081]To perform RT-PCR, 1 μl (100 pg) of lambda DNA, 1 μl (10 pmol) of each of the above primers and probe, 6.45 ul of 3.1× qPCRMix and distilled water were mixed in a PCR tube to have a final volume of 20 μl.
[0082]It was observed that a typical pattern of FAM signals induced ...
example 2
pending on Structural Features of 5′-End of DSP Probes
[0083]In this example, it was confirmed whether change of 5′ nuclease activity of DNA polymerase depends on 5′-end structures of DSPs. No flap structure (Mis+0) and flap structures consisting of one base (Mis+1), two bases (Mis+2) or three bases (Mis+3) at 5′-end structure of probes were used in this example.
*PrimersForward primer:5′-TAMRA-gccgcgctggatgaactgatac-3′Reverse primer:5′-cggcctgaacagtgagcgaag-3′*ProbesMis + 0:5′-ccggtatcagttcatccagcgc-FAM-3′Mis + 1:5′-tccggtatcagttcatccagcgc-FAM-3′Mis + 2:5′-atccggtatcagttcatccagcgc-FAM-3′Mis + 3:5′-catccggtatcagttcatccagcgc-FAM-3′
[0084]* RT-PCR reaction condition:
[0085]95° C., 5 min. (1 time)
[0086]95° C., 15 sec-60° C., 30 sec-72° C., 30 sec (40 cycles)
[0087]* Composition for RT-PCR reaction
[0088]To perform RT-PCR, 1 μl (100 pg) of lambda DNA, 1 μl (10 pmol) of each of the primerset and the probes as above, and 6.45 μl of 3.1× qPCRMix and DW were mixed in a PCR tube to have a final vo...
example 3
ing Probes Having 5′-Flap Structures of Various Forms
[0090]In this example, in order to confirm inhibition of FEN activity by 5′-end structure in Example 2, change of 5′ nuclease activity of Taq DNA polymerase according to the 5′-end structures of DSP, SYBR Green probe and external TaqMan probe were confirmed.
[0091]The primers, probes and RT-PCR conditions in this example were as follows.
*Primers and probes for DSP test (FIG. 4a)Forward primer:5′-TAMRA-gccgcgctggatgaactgatac-3′Reverse primer:5′-cggcctgaacagtgagcgaag-3′*ProbesMis + 0:5′-ccggtatcagttcatccagcgc-FAM-3′Mis + 1:5′-tccggtatcagttcatccagcgc-FAM-3′Mis + 2:5′-atccggtatcagttcatccagcgc-FAM-3′*Primers and probes for SYBR Green probe test(FIG. 4b)Forward primer:5′-gccgcgctggatgaactgatac-3′Reverse primer:5′-cggcctgaacagtgagcgaag-3′*ProbesMis + 0:5′-ccggtatcagttcatccagcgc-3′Mis + 1:5′-tccggtatcagttcatccagcgc-3′Mis + 2:5′-atccggtatcagttcatccagcgc-3′*Primers and probes for external TaqMan probe test(FIG. 5a)Forward primer:5′-taccggggt...
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