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讼郑
7 years ago
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1 Real-Time PCR 原理及應用研究部黃欣儀

2 基本原理 : Real-Time PCR 隨著 PCR 反應循環逐次增加時, 目標 DNA 也隨之快速增幅, 此時 PCR 反應液反應中之特定的螢光物質可與目標 DNA 結合而產生螢光, 經由儀器偵測後即時將訊號資料呈現並計算

3 基本原理 : 目標 DNA 起始的量愈多, 其螢光值愈早達到偵測之閥值 (Threshold), 此時所對應的循環數 (cycle) 稱之為 Ct 值因此, 目標 DNA 的濃度與 Ct 值成反比關係依據連續稀釋標準樣品的 Ct 值及已知濃度, 可得標準曲線, 根據此標準曲線可以推算樣品的起始濃度, 達到定量測試之目的

4 Real-Time PCR 原理 A. 螢光化學原理 B. 螢光強度計算原理 C. 數據分析 Real-Time PCR 應用 A. 絕對定量 Absolute Quantification B. 相對定量 Relative Quantification C. 基因型鑑定 SNP genotyping assay D. 微小 RNA 偵測及定量

5 Real-Time PCR 螢光化學原理雙股 DNA 嵌合螢光染劑 SYBR Green dye

6 SYBR Green dye a highly specific, double-stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles. 優點 : 不需額外合成特殊引子, 方法簡單成本低, 可作 melting curve 分析缺點 : 若 PCR 過程中有或有 primer dimer 或有非專一性 PCR 產物均會影響判讀

7 Real-Time PCR 螢光化學原理特定序列螢光探針 TagMan probe dual labeled probe hydrolysis probe Molecular beacons FRET-Dual labeled probehybridization probe

8 TagMan probe dual labeled probe hydrolysis probe 5 -end fluorophore:. FAM, TET 3 -end quencher: TAMRA, MGB In Gene expression assays Pharmacogenomics Human Leukocyte Antigen (HLA) genotyping Determine the viral load in clinical specimens (HIV, tuberculosis, Hepatitis) Bacterial Identification assays DNA quantification SNP genotyping microrna quantification 優點 : 專一性佳缺點 : 需額外合成特殊引子, 成本高, 無法作 melting curve

9 Molecular beacons Loop 部分為專一性序列優點 : 專一性佳, 可作 melting curve 缺點 : 需額外合成特殊引子, 成本高

10 FRET-Dual labeled probe-hybridization probe 3-5bp 優點 : 專一性佳, 可作 melting curve 缺點 : 需額外合成特殊引子, 成本高

11 Real-Time PCR 螢光強度計算原理 Amplified curve: 4 phases 線性曲線 plateau phase 對數曲線 linear phase plateau phase Basal phase linear phase Exponential phase Exponential phase Basal phase Ct threshold

12 數據分析 : 如何定義閥值 (Threshold) Sigmoidal curve: 4 factors X n : 在 n 次循環下之產物分子數 X 0 : 起始分子數 E x : 擴增效率 n: 循環數 Semi-log

13 Real-Time PCR 應用絕對定量 Absolute Quantification 用於確定未知樣本中某個核酸序列的絕對量值, 也就是 copy numbers Ct=-klogx+b

14 相對定量 Relative Quantification 用於測定樣本中之目標基因 (target gene) 在實驗組及對照組中表達的相對變化在比較之前, 同一個樣本中的目標基因需用內部對照基因 (endogenous control gene) 先做校正 (normalization) Ct ref Ct target 實驗組對照組

15 相對定量 Relative Quantification Target Endogenous Ct samplegene control Ct control (Control)

16 基因型鑑定 Single nucleotide polymorphisms genotyping assay 需用兩個帶有不同螢光物質, 並針對單一鹼基變異而能辨識之探針, 在 PCR 反應完成時收集數據, 可用於檢測變異每個反應中需包括兩個引子和探針, 可將序列放大並偵測產物序列上單核苷酸多形性位點上出現兩個可能的變異基因型目標序列實際量不確定

17 Allele T G A A Allele G T Quencher C C C/C A/A A/C

18 Allelic Discrimination plot FAM C/C A/C A/A VIC

19 One Step Real time PCR method RT Melting curve 48:00 30:00

20 High Resolution Melt (HRM) Novel, homogeneous, post-pcr method, enabling genomic researchers to analyze genetic variations in PCR amplicons. Beyond the power of classical melting curve analysis in much more detail and with much higher information yield than ever before. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes can be readily identified.

21 HRM Applications Mutation discovery (gene scanning) Screening for loss of heterozygosity DNA fingerprinting SNP genotyping Characterization of haplotype blocks DNA methylation analysis DNA mapping Species identification Somatic acquired mutation ratios HLA compatibility typing Association (case/control) studies Allelic prevalence in a population Identification of candidate predisposition genes

22 Basic primer Genotyping assay Homozygous WT Homozygous Mut Heterozygous Sequence specific probe

23 T m differences between homozygotes are big enough, these can be displayed by omitting the temperature-shift step.

24 微小 RNA 偵測及定量

25 微小 RNA 偵測及定量

26 目標模板 (Template) 選擇之原則 Design amplicon to be bp. Shorter amplicons are typically amplified withhigher efficiency. An amplicon should be at least 75 bp to easily distinguish itfrom any primer-dimers that might form. Avoid secondary structure if possible. Avoid templates with long (>4) repeats of single base. Maintain a GC content of 50 60%.

27 引子設計之原則引子設計原則 Length in 20~40 bp Design primers with a GC content of 50 60% Maintain a melting temperature (Tm) between 50ºC and 65ºC. Avoid secondary structure Avoid repeats of Gs or Cs longer than three bases Place Gs and Cs on ends of primers. Number of Cs> Gs Avoid primer dimer & hairpin form (<5 complementarity, low G-C)

28 ABI Primer Express 3.0

30 醫院現有之機型研究部八樓 ABI 7900HT 子宮頸癌專題研究室 StepOnePlus Real-Time PCR System

31 感謝聆聽

SDS 1.3

SDS 1.3 Applied Biosystems 7300 Real-Time PCR System (With RQ Study) SDS 1.3 I. ~ I. 1. : Dell GX280 2.8GHz with Dell 17 Flat monitor 256 MB RAM 40 GB hard drive DVD-RW drive Microsoft Windows XP Operating System