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LIFE SCIENCE TECHNOLOGIES: The Digital PCR Revolution

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LIFE SCIENCE TECHNOLOGIES:

The Digital PCR Revolution

林俊叡 梁世融 魏晧軒 潘世璋 鄭家凱 姜智偉 楊筌凱

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DNA 雙股螺旋

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核甘酸

核甘酸 (Nucleotide) 為核酸分子構成單元

核甘酸包含:

五碳糖 ( 去氧核糖 , deoxyribose)

磷酸基 (phosphate group)

含氮鹼基之一 (A 、 G 、 C 、 T 、 U)

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DNA 與 RNA

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轉錄與轉譯作用形成蛋白質

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轉錄與轉譯作用形成蛋白質

PCR

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蛋白質的結構

• 1. 一級結構 (primary structure)

• 2. 二級結構 (secondary structure)

• α 螺旋 (α-helix)

• β 摺疊 (beta sheet)

• 3. 三級結構 (tertiary structure)

• 4. 四級結構 (quaternary structure)

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一級結構 (primary structure)

• 肽或蛋白質的氨基酸序列(或殘基序列)被稱為一級結構。

• 一級結構上的胺基酸間可交互作用,利用醯胺鍵上的 C=O 鍵與胺 基形成氫鍵。

(9)

二級結構 (secondary structure)

• 多肽因連接各胺基酸的肽鍵 (peptide bond) 間產生氫鍵,而形成重複 出現的特殊結構 : 1. α- 螺旋 2. β- 褶片

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三級結構 (tertiary structure)

• 指已具有二級構造的多肽,因胺基酸側鏈間的交互作用而折疊扭 轉成特有的緊密立體形狀

• 三級結構描述這些 domain 的關係以及蛋白質折疊讓序列中距離 遙遠的氨基酸互相靠近的途徑,以及使其構形穩定的鍵結。

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四級結構 (quaternary structure)

• 當具有生物功能的蛋白質是由兩條或兩條以上的多肽 ( 次單元 ) 組成時,次單元在立體空間的相互關係

• 形成四級構造的優點

• 增加結構安定性

• 遺傳物質能有效利用

• 形成功能或活性部位

• 調節與協同效應

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蛋白質之重要性

1. 不同蛋白質所具有之獨特序列,除了蘊含千變萬化的結構之外,也賦予 其在各種生命現象中背負繁複但重要的使命,例如

基因的複製

細胞週期的調控

養分的運送

代謝反應

訊息傳導等

2. 蛋白質透過彼此的交互作用,形成綿密的蛋白質網絡,以維持生命體的 正常運作,因此當某個蛋白質遭致先天突變或後天修飾而失常,致使原 來應有的功能減損或喪失,蛋白質網絡的失衡,牽一髮動全身的結果,

將造成細胞病變而產生疾病。

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蛋白質變異的原因

1. 基因變異 2. 結構變異

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蛋白質變異的原因

1. 基因變異 2. 結構變異

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基因變異

1. 導致蛋白質的生合成、傳輸、穩定度乃至於酵素活性受到影響,使得原來 應有之功能喪失或減損

2. 基因變異的種類,依據現在分子層面的瞭解可分成:

a) 基因中單一核甘酸序列的變異

b) 基因中小片段重複序列的拷貝數目改變 c) 一基因內插入一大段外來的 DNA 片段

3. 染色體結構的變異

a) 缺失( deletion ) - 一至多個 DNA 序列消失

b) 反轉( inversion ) - 基因在染色體上的排列方式前後倒置 c) 位移( translocation )

d) 重複( duplication )

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基因變異 cont

• 基因發生核甘酸序列的變異時,經由轉錄,這些變異的核甘酸序 列被忠實的轉錄到 mRNA 的核甘酸序列上,因此轉譯出的胺基酸 種類也就不同,不同的胺基酸造成蛋白質特性的改變

• 自閉症 - 患者有一種蛋白質變異,這種蛋白質原用來幫助腦細胞 經神經通路「突觸」 (synapses) 轉換資料,

導致患者腦細胞間的溝通程度,縮減到僅有 一般人的十分之一

(18)

基因變異 cont

• 癌症 - 控制調節細胞分裂的機制相關基因發生變異所造成

• 蠶豆症、苯酮尿症、鎌刀型貧血症 - 體染色體隱性遺傳疾病

• A 型血友病、色盲 -X 染色體聯鎖隱性遺傳疾病

• 低磷酸鹽性佝僂症 -X 染色體聯鎖顯性遺傳疾病

• 唐氏症 - 染色體結構變異,大多數為第 21 號染色體多了一條

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蛋白質變異的原因

1. 基因變異 2. 結構變異

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蛋白質結構變異

• 蛋白質結構本身受到構型重整 (conformational rearrangement) 或錯誤折疊 (misfolding) 的影響,而致使蛋白質有自發性聚合

(self-association) 的現象 ( 如 β-linkage) ,造成聚合之蛋白 質在細胞內產生組織堆積 (tissue deposition) 或內含體 (inclu sion body) 而致病

• 例如常見於各大媒體報導的神經退化性疾病阿茲海默症 (Alzheim er’s disease) 、巴金森氏症 (Parkinson’s disease) ,與為 人談之色變並影響農業經濟甚巨的狂牛症 (Bovine spongiform e ncephalopathy

(21)

DNA hydrogen bondings

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Primer: A short segment of DNA sequences Examples:

Primer 1: ATTGC

Primer 2: GGTGCA

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Primer and DNA binding Examples:

Primer 1: ATTGC

DNA 1 : TAACGTCGATGCCTTAG

Primer 2: GGTGCA

DNA 2 : CCACGTTATCCGTAGCGTC

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Primer and DNA un-binding Primer 1: ATTGC

DNA 1 : TAACGTCGATGCCTTAG 3(A-T) pairs and 2(G-C) pairs:

12 hydrogen bondings Primer 2: GGTGCA

DNA 2 : CCACGTTATCCGTAGCGTC 2(A-T) pairs and 4(G-C) pairs:

16 hydrogen bondings

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PCR: Polymerase chain reaction

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PCR 的原理與方法三

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PCR 的原理與方法四

(28)

Real-Time PCR

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Real-time PCR

• Fluorescent reporter probe m ethod

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Real-time PCR

due to variations in reaction kinetics,different q uantities of PCR product by the plateau phase o f the reaction

it will be more precise to take measurements d uring the exponential phase

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Advantage

• Reduce the experiment time

• more sensitive & accurate

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Ct value

The PCR cycle at which the sample reaches a f uorescent intensity above background is the Cy cle Threshold or Ct

making it possible to determine the starting co ncentration of nucleic acid

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Digital PCR

Present By 生醫電資所 姜智偉

Bert Vogelstein, Kenneth W. Kinzler Proc Natil.Acad.

Sci. 9236-9241. 1999

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Introduction

• Cancer

• Genecell proliferationcancer,

• Curable (minor tissue injury) curable(major tissue injury)Non-curable.

• Virus

• HIV (AIDS) with treatment Difficult to detect HIV virus.

• Monitor virus mutation and latent virus

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Why we need to develop Digital PCR???

• Detection small tumor cell from large amount of normal cell (Looking a needle in the haystack)

• Urine

• Stool

• Blood

• Not contamination by normal cell after amplification

• Absolute quantification

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Digital PCR method

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Special probe design

• Stem-loop formation

• Temperature change shape cha nge

• Flourescence energy was inversel y proportional to 6 power of dista nce between two point.

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Probe and target DNA

• MB-Green attach to Mutation site DNA

• MB-Red attach to normal site

• Successful amplification Wild type PCR both MB-Red and MB-Gree n gives light

• Successful amplification of Mutati on type PCR on ly MB-Red gives light

(39)

Dilute and PCR amplification

• Try to Dilute DNA to ½ copy per w ell

• Repeat temperatrure control

• PCR amplification

• Probe detection

• Read the data (Red/Green ratio)

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C-Ki-Ras mutation and R/G ratio

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C-Ki-Ras from tumor cell

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Stool sample for C-Ki-Ras (Colorectal canc

er)

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Characteristics of Digital PCR

• Prevent contamination

• Dilution and Partition to ultra small molecule

• In situ PCR amplification in each well

• Absolute quantification

• Simply detection of Mutation/Wild type, not every Mutation type

• Poisson distribution

• Quality control

• Thermal cycle

• Special primer design

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Video Demonstration

Introduction o Digital PCR

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Thank you

(46)

Comparison between Tradition al PCR and Real-time PCR and

Digital PCR

楊筌凱

(47)

Three phases of PCR

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Detection area

(49)

Cycle threshold of real-time PCR

(50)

Digital PCR works by partitioning a sample into many individual real-time PCR reactions, some portion of these reactions contain the target molecules(positive) while others do not (negative).

The fraction of negative answers is used as reference.

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參考文獻

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