第讲基因工程的主要技术分析.ppt

生物技术核心课程 A 重组DNA技术的理论基础 B 基因的分子生物学 C 基因工程的基本原理 第一讲 基因工程概论 第二讲 基因工程的主要技术 第三讲 基因工程的酶学基础 第四讲 基因克隆的载体与受体 第五讲 目的基因的克隆与分离 第六讲 克隆基因的检测与功能鉴定 第七讲 克隆基因的表达与产物纯化 第八讲 基因表达的调控 三篇重要论文 DNA双链非特异性内嵌染料（SYBR GreenⅠ）能特异性地结合到双链DNA上，而不结合到单链DNA上，并且结合后的染料的发光强度会明显增加，没有结合的染料在溶液中只能发出很微弱的荧光。 扩增序列特异性检测方法是在PCR反应中利用标记荧光染料的基因特异寡核苷酸探针来检测产物，它又可分为直接法和间接法。 间接法（双标记探针Taqman Probe） : PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针，该探针为一寡核苷酸，两端分别标记一个报告荧光基团和一个淬灭荧光基团，此时5′端荧光基团吸收能量后将能量转移给临近的3′端荧光淬灭基团（发生荧光共振能量转移，FRET），因此探针完整时，检测不到该探针5′端荧光基团发出的荧光。Fluorescent probesFluorescent probes are pieces of DNA complimentary to your gene of interest that are labeled with a fluorescent dye.The simplest and most commonly used type of probe is the Taqman-type probe. These probes are labeled with a fluorescent reporter molecule at one end and a quencher molecule (capable of quenching the fluorescence of the reporter) at the other. Hence under normal circumstances the fluorescent emission from the probe is low. Howeverduring the PCR the probe binds to the gene of interest and becomes cleaved by the polymerase. Hence the reporter and quencher are physically separated and the fluorescence increases. 本质上是一种标记荧光的发夹探针，当探针分子呈发夹结构时，结合在其两端的荧光基团距离上接近，使得产生能量转移效应，而不发生荧光。当互补序列出现时，探针与DNA杂交，探针转变成一个开放的结构，呈线性，报告荧光基团与淬灭荧光基团彼此在空间上产生足够的分离，荧光基团脱离了淬灭基团的影响，从而产生可被检测到的荧光 。 Another commonly used type of probe is the “molecular beacon”. Again these are small pieces of DNA complimentary to your gene of interest labeled with a fluorescent reporter and a quencher molecule on opposite ends. These probes are designed to fold on to themselves to bring the reporter and quencher in to closer proximity and minimize fluorescent emission. However, when the probe binds to the gene of interest the probe takes up a linear confirmation and the reporter and quencher are separated.This results in the desired increase in fluorescence. Molecular beacon probes are not cleaved by the polymerase but are simply “knocked off” again. 紫外光谱分析法的原理基于DNA（RNA）分子在260nm处有特异的紫外吸收峰且吸