DNA DNA cloning DNA Recombinant DNA Libraries DNA

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

DNA克隆的一般流程 1. 分离DNA 2. 限制性内切酶酶切DNA 3. 连接于克隆载体构建重组DNA分子 Cloning vector = artificial DNA molecule capable of replicating in a host organism (e. g. , bacteria). 4. 重组分子的转化

DNA的提取 • SDS (detergent) to break up cell membrane and organelles. • Salt (Na. Cl) lyses cells and binds the DNA strands together. • Proteinase K to digest proteins bound to DNA. • Ethanol (Et. OH) to precipitate and wash DNA. • Water to resuspend and store DNA.

分子克隆载体 载体的类型有: 1. 质粒 Plasmid cloning vectors 2. 噬菌体 Phage cloning vectors 3. 黏粒 Cosmid cloning vectors 4. 穿梭载体 Shuttle vectors 5. 酵母人 染色体 Yeast artificial chromosomes (YACs) 6. 细菌人 染色体 Bacterial artificial chromosomes (BACs)

粘粒克隆载体 1. 兼有质粒和噬菌体的特性. 2. 人 合成的. 3. 复制起点（ORI）. 4. 选择性标记（ amp. R）. 5. 多克隆位点. 6. Phage cos site permits packaging into phages and introduction to E. coli cells (useful for 37 -52 kb).

细菌人 染色体（BAC） 1. 可克隆200 kb的外源DNA片段. 2. 通常用于染色体大片段DNA的测序. 3. 宿主细胞为细菌，很象常规的细菌质粒载体 4. 特征性结构有： v Origin (ori) sequence derived from an E. coli plasmid called the F factor. v Multiple cloning sites (restriction sites). v Selectable markers.

Ø Plasmid <10 kb Ø 噬菌体 0~23 kb Ø 粘粒 33~45 kb Ø BAC ~100 kb Ø YAC 300 kb~1. 2 Mb

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

基因组DNA文库的构建 完全酶切法 1. 2. Produces a large number of short DNA clones. Genes containing two or more restriction sites may be clones in two or more pieces. 机械切割法 1. 2. Produces longer DNA fragments. Ends are not uniform, requires enzymatic modification before fragments can be inserted into a cloning vector. 部分酶切法 1. 2. 3. 4. Cut at a less frequent restriction site and limit the amount and time the enzyme is active. Results in population of large overlapping fragments. Fragments can be size selected by agarose electrophoresis. Fragments have sticky ends and can be cloned directly.

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

Kary B. Mullis Polymerase Chain Reaction (PCR) April 1983 1993: Nobel Prize for Chemistry 1989年美国《Science》杂志列PCR 为十余项重大科学发 明之首, 比喻 1989年为PCR爆炸年, Mullis荣获 1993年度诺 贝尔化学奖。

PCR技术原理 35 94° 30 s 94° 3 min 55° 30 s 72° 40 s 72° 10 min

基因 程技术及原理 DNA克隆技术 DNA cloning DNA文库构建及筛选技术 Recombinant DNA Libraries DNA杂交技术 DNA hybridization PCR技术 Polymerase Chain Reaction (PCR) DNA测序技术 DNA sequencing

Radio-labeled dd. NTPs (4 rxns) Sequence (5’ to 3’) G G A T A A C C T G T Short products Long products

“virtual autorad” - real-time DNA sequence output from ABI 377 1. Trace files (dye signals) are analyzed and bases called to create chromatograms. 2. Chromatograms from opposite strands are reconciled with software to create doublestranded sequence data.