biorad定量pcr說明書(編輯修改稿)
2025-06-19 09:01 本頁面
【文章內(nèi)容簡介】 ? Identify SubOptimal aspects of assay Optimize further with thermal gradient, etc. Cleavage Probes (TaqManTM) 5’ Q F Taq 5’ q 3’ l 5’ ? 雜交探針的互補(bǔ)區(qū)在引物間 ? 5’端連有一個熒光 reporter, 通常是熒光素 ? 3’端連有一個 Quencher,通常是 TAMRA ? 熒光素被 488 nm光激發(fā)，發(fā)射光為 520 nm ? 520 nm 光能激發(fā) TAMRA， 發(fā)射光為 570 nm TaqMan Cleavagebased assay: TaqManTM 5’ 5’ 3’ 3’ Thermal Stable DNA Polymerase Primers Add Master Mix and Sample Denaturation Annealing Reaction Tube Taq l R Q Probe R Q 5’ 3’ R Q Extension Step 5’ 3’ 1. Strand Displacement Taq Q R 5’ 3’ Q Taq R 5’ 2. Cleavage 3. Polymerization Complete 5’ 3’ Taq R 5’ 4. Detection 5’ 3’ Taq R 5’ l R Cleavagebased assay: TaqManTM Cleavage Probes (TaqManTM) Advantages! ? Generates a robust cumulative fluorescence signal ? Simple to design and synthesize pared to other hybridization probes (. beacons) ? Ideal approach for multiplex assays ? SNP (Single Nucleotide Polymorphism) assay possible However… ? More expensive than DNA binding dyes Molecular Beacons R Q Molecular Beacon 5’ 3’ R Q ? 探針有核心的雜交區(qū) ? 探針有自互補(bǔ)末端 ? 在未雜交時探針呈發(fā)夾狀 ? 報道子 , 熒光素在探針的 5’ 端 ? 猝滅子在探針的 3’ 端 分子 Beacons ? 當(dāng)探針是發(fā)夾結(jié)構(gòu)時，在報道子和猝滅子間有直接的能量轉(zhuǎn)移 Beacon 構(gòu)造 Molecular Beacons Advantages! ? Good for SNP (Single Nucleotide Polymorphism) detection ? Multiplex assays possible However… ? Molecular Beacons Are DIFFICULT to Design and Synthesize ? Does NOT generate a cumulative fluorescence signal, much weaker signal than the 5’ Nuclease Assay (Cleavage probe) ? Expensive! A Few Words about Probes… ? Hybridization oligos should have Tms 6 – 12 degrees higher than the associated primers. ? Avoid secondary structure in the plementary region of the probe. ? Avoid G’s at the 5’ end of the probe sequence. ? Use oligo analysis tools to check probe for: Dimerization Secondary Structure Cross Reactivity with Primers ? Each method has advantages and disadvantages ? BioRad RealTime Instrumentation is equipped to handle all chemistries ? One method may be more appropriate for an application over another Which Method to Use? Outli
試題試卷相關(guān)推薦
鄂ICP備17016276號-1