原理应用探针设计和应用实例.pptVIP

荧光定量PCR仪应满足的要求 光源的光谱范围宽广，能激发不同的荧光染料 能分开不同荧光素的激发光与发射光波长 能检测的灵敏度与动态检测范围 准确的温度控制和良好的孔间温度均一性. 荧光定量PCR原理及应用 荧光定量PCR实验引物/探针设计及实验条件优化 Stratagene Mx3000P荧光定量PCR仪 QPCR Assay Optimization Design Synthesis Test oligos Optimization Primers Probe Enzyme,dNTP, Mg++ Serial dilution Run and Analysis Target/Amplicon Choice Target size: 50-150 bp for dual-labeled probes (Taqman) 200-400 bp for SYBR If using a cDNA template, span introns to avoid genomic DNA contamination. Try to avoid repetitive or highly structured regions (CpG islands and control regions at the 5’end tend to form complex structures). QPCR Primer Properties Use the same target annealing temperature for all your primers to favor multiplexing in the future and run single thermal profile Typically 55-60°C (with 1°C temp difference) Avoid G/C clamps at the 3’end. If requested by the software, use 100 mM monovalent cation and 5 mM Mg++ for probes, 2.5 mM for SYBR. Always BLAST your primers. Dual-labeled Probe Design Tm 10°C higher than primers. 35-65% G/C;more Cs than Gs. Avoid runs of 3+ of the same nucleotide, especially Gs. 5’ base ? G. 3’ end of primer and 5’ end of probe on same strand between 1-15 bp distance. Test that primers and probe are not complementary to each other. Reporter-Quencher Pairs Dye Quencher FAM BHQ-1/TAMRA HEX/JOE BHQ-2 Texas Red/ROX BHQ-2 Cy5/Quasar670 BHQ-2 ( or -3) Oligo Synthesis Primers: Use desalted primers for sequence-specific detection. SYBR Green primers should be HPLC-purified. Aliquot stock solution of 20x (as determined by the optimization step), typically 6 - 18μM each oligo. Test primers before ordering probe. Probe: Ensure matching fluorophore/quencher set. Double-HPLC purified (purification also OK). Aliquot working stock at 2 - 4μM (20x). Initial Primer Testing Primers: PCR reaction and electrophoresis. SYBR Green run and dissociation analysis. Melting Curve Analysis Good Probe Performance dRn = 0.1 to 0.9 Poor Probe Performance dRn = 0.1 to 0.4 QPCR Assay