R F L P
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Transcript of R F L P
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R F L PR F L P
限制性片段长度多态性
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R F L PR F L P
Restriction Fragment Length Polymorphism
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是随着生物技术若干领域的发展而产生的一种分子标记技术
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一. 限制性内切酶( restric
tion endonuclease )
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1. 1. 简 介简 介
限制性内切酶是一类具有特殊功能的核酸切割酶,不同的内切酶可辨认并作用于特异的 DNA 序列,并将 DNA切断
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I 型 酶 :
如酶的识别位点上两条 DNA 链均未甲基化,就行使内切酶功能,但切割点不在识别位点,而在 1kb (不少于 400bp )或 nkb( 最多 7kb) 处随机切割;如一条 DNA 链是甲基化,就行使甲基化酶功能,使另一条链甲基化;如两条链均已甲基化,则酶与 DNA 链解离。
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III 型 酶
与 I 型酶特性类似,也有甲基化功能。不同的是它能在 DNA 链上的特异位点切割,其切割位点在识别位点以外,对基因工程的意义也不大。
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II 型 酶
就是通常指的 DNA 限制性内切酶, II 型限制酶分子量小、仅需 Mg2 +作为催化反应辅助因子。它们能识别双链 DNA的特异顺序,并在这个顺序内进行切割,产生特异的 DNA片段。
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II 型限制性内切酶的最大特点是识别 DNA 特异顺序并进行切割,因而在基因重组、 DNA 序列分析、基因组甲基化分析及基因物理图谱等技术中广泛应用。
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2. 2. 性质和特点:性质和特点:
•分子大小 分子大小 2020 -- 100kdal100kdal•作用条件 对离子强度要求高作用条件 对离子强度要求高•识别顺序 大多识别顺序 大多 44 -- 6bp 6bp 迴文迴文
结构结构•切口 就在识别顺序内切口 就在识别顺序内
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同工异源酶 ( isoschizomer )
不同来源分离出来,但具有相同识别顺序,切割 DNA 方式可以相同,也可以不同。
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Sma I Xma ISma I Xma I
C C C G G G C C C G G GC C C G G G C C C G G G G G G C C C G G G C C CG G G C C C G G G C C C
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同尾酶( subset )
不同的酶但切割后突起的核苷酸序列相同,可相互连接,重组后可能不被原先酶识别。
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BamH I: BamH I: GGATCCGGATCC CCTAGGCCTAGG Bgl II:Bgl II: GGGATCTGATCT AGATCT AGATCT CCTAGCCTAGAA TCTAGATCTAGA
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远距离裂解酶( distant cleavage )
识别位点与切割位点不一致,切割点在识别位点旁 10bp 左右,如 Mb2 识别顺序 GAAGA, 切割位点在下游第 8 个 bp 。
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可 变 酶
–识别顺序中 1 个或几个核苷酸是可变的,识别顺序 >6bp 。
–如 Bgl I 识别 GCC(N5)GGC, 其 N5 为 5 个可变碱基。
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3. 内切酶活性定义:在适当反应条件下, 1 小时内完
全酶解 1g 特定 DNA 底物所需要的限制性内切酶的量,为一个活性单位。
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二、 DNA 限制性内切酶酶
切图谱
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限制性内切酶图谱:也称 DNA 物理图谱,是 DNA 链上某些限制性内切酶酶切位点的图谱,可作为 DNA 片段的特殊标记。是 DNA 特征的证据。
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–识别 4 个核苷酸的内切酶,在 DNA 链上出现机率为 :
1 / 44 = 1 / 256bp
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–识别 6 个核苷酸的内切酶,在 DNA 链上出现机率为 :
1 / 46 = 1 / 4.1kb
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–识别 8 个核苷酸的内切酶,在 DNA 链上出现机率为 :
1 / 48 = 1 / 65.5kb
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DNA 限制性内切酶酶切图谱的建立
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AA 、部分酶解法:、部分酶解法:
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①① 完全酶解 完全酶解 3 3 ,, 22 , , 1 kb1 kb
②② 部分酶解 部分酶解 66 ,, 55 ,, 44 ,, 33 ,, 2, 1 k2, 1 k
bb
③③ 分析 分析 5=3+2 4=3+1 35=3+2 4=3+1 3 在中间在中间
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B.B. 两种以上限制性内切酶交叉酶解法:两种以上限制性内切酶交叉酶解法:
–总长 总长 EcoRⅠ HpaⅡEcoRⅠ HpaⅡ
– 3kb 1.73kb 1.7 ,, 0.90.9 ,, 0.4 1.60.4 1.6 ,, 1.41.4
– EcoRⅠ+ HpaⅡ EcoRⅠ+ HpaⅡ
– 1.2 , 0.9 , 0.5 , 0.41.2 , 0.9 , 0.5 , 0.4
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总长 总长 EcoRⅠ HpaⅡEcoRⅠ HpaⅡ3kb 1.73kb 1.7 ,, 0.90.9 ,, 0.4 1.60.4 1.6 ,, 1.41.4 EcoRⅠ+ HpaⅡ EcoRⅠ+ HpaⅡ 1.2 , 0.9 , 0.5 , 0.4 1.2 , 0.9 , 0.5 , 0.4
–分析: ①分析: ① HpaⅡHpaⅡ 一个切点,可肯定一个切点,可肯定
②② EcoRⅠ1.7 kbEcoRⅠ1.7 kb 不在末端,在中间不在末端,在中间
③③ EcoRⅠ 0.9EcoRⅠ 0.9 和和 0.4 kb0.4 kb 都在末端都在末端
–④④ 逻辑判断以上二片段在哪一边末端逻辑判断以上二片段在哪一边末端
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C.C. 同位素末端标记,部分酶解法同位素末端标记,部分酶解法
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三、 RFLP 的检测
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1 、 RFLP 产生的原因
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在一个特定基因座位上,存在着两个或两个以上的等位基因,其中任何一个在人群中频率不低于 10% ,这就是多态性。
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但现在认为不同个体中同一位点上 DNA 核苷酸序列产生变异,且在人群中发生频率大于 1% ,就可认为是多态性。
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SNP (单核苷酸多态性),由单个核苷酸变异引起的 DNA 顺序的多态性,估计人类基因组中存在约几百万个,约每 1000个 bp 左右就有一个。
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–如在限制性内切酶酶切位点发生核苷酸的变异(或多态性),就会产生 RFLP ,除 SNP 外,还包括缺失、重复、插入、基因重排以及串联重复序列拷贝数的变异。
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2. 检测方法
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(1). 酶切电泳法:抽提 DNA 或 PCR扩增后的 D
NA经内切酶酶切 , 然后在琼脂糖凝胶电泳分析,适合较小分子的 DNA 分析
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(2). Southern Blot:
DNA 先酶切,电泳分离,变性后转移到尼龙膜或硝酸纤维膜,然后与同位素标记的探针杂交,最后做放射自显形,就可检测出多态性条带。
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四. 应 用
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1. 鉴定是否是需要的 DNA 片段
酶切和电泳
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2. 建立基因组图谱
用作多片段的拼装
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3. 遗传性疾病的研究和诊断
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( 1 ) . 研 究
根据多态性片段寻找与疾病相关的基因。
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(2). 诊 断
– 直接检测– 连锁分析
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苯丙酮尿症连锁分析苯丙酮尿症连锁分析 父 (杂合子) 母(杂合父 (杂合子) 母(杂合
子)子) EcoR V 30kb /25kb 30kb/25kb EcoR V 30kb /25kb 30kb/25kb
患儿(纯合子) 胎儿(?)患儿(纯合子) 胎儿(?) 30kb/30kb 30kb/25kb30kb/30kb 30kb/25kb
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4. 疾病易感或抵抗基因的 检测
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HLA DQ HLA DQ 分子分子 Asp57 Asp57 与胰与胰島素依赖性糖尿病(島素依赖性糖尿病( IDDIDDMM)易感性有关。)易感性有关。
HLA DR2HLA DR2 与与 IDDM IDDM 抵抗有关。抵抗有关。
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5. 器官移植配型
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6. 病原体的分型
HPV 6 , 11 型诱发湿疣 HPV 16 , 18 型诱发生殖道癌症
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7. 药物遗传学和药物基因组学
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药物遗传学主要研究遗传因素对药物代谢和药物反应的影响,尤其在异常的药物反应中的作用。
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药物基因组学不仅仅研究与药物代谢有关的基因,还要研究所有与药物作用有关的基因,这些基因在不同个体中的多样性,以及这些多样性对药物的疗效、毒性或副作用等的影响。
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8. 其 他
– 单卵双生或双卵双生的诊断– 个体识别 – 人类学研究– 动植物分类等等。