DNA DNA cloning DNA Recombinant DNA Libraries DNA


































































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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.
Recombinant DNA OVERVIEW OF RECOMBINANT DNA TECHNOLOGY Recombinant
Recombinant DNA cloning technology Chapter 7 DNA cloning
Cloning Vectors Gene Cloning Cloning a definition From
BASICS OF MOLECULAR CLONING Defining Cloning Cloning is
Cloning into Plasmids Restriction Fragment Cloning PCR Cloning
CLONING TECHNIQUE What is cloning Cloning is a
Unit Eight Cloning What is cloning Cloning is
Cloning 3 Types of Cloning Transgenic gene cloning
Unit Eight Cloning What is cloning Cloning is
BIOTECHNOLOGY Recombinant DNA Recombinant DNA a fragment of
DNA recombinant technology Definition of recombinant DNA technology
Recombinant DNA Technology Recombinant DNA Protocols that transfer
Recombinant DNA Technology restriction enzymes making recombinant DNA
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Lecture 7 Recombinant DNA OVERVIEW OF RECOMBINANT DNA
RECOMBINANT DNA TECHNOLOGY RECOMBINANT DNA TECHNOLOGY Scientists have
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libraries Cosmid libraries BAC libraries Cosmid libraries BAC
libraries Cosmid libraries BAC libraries Cosmid libraries BAC
libraries Cosmid libraries BAC libraries Cosmid libraries BAC
Recombinant DNA Bacterial Transformation Student Instructions Transformation Recombinant
RECOMBINANT DNA Introduction Definition Steps Applications INTRODUCTION Recombinant
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Cloning Recombinant DNA Multistep Process Produce fragments of
1 I Recombinant DNA Gene cloning F II
Molecular Cloning or Genetic Engineering or Recombinant DNA
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Cloning a DNA segment from lambda bacteriophage Recombinant
Principles of cloning vectors and cloning strategies DNA
Recombinant DNA Technology BTEC 3301 DNA Libraries l
RECOMBINANT LH RECOMBINANT HCG AND GNRH AGONIST TO
Chapter 13 An Introduction to Cloning and Recombinant
Cloning sequencing and recombinant production of Sin a
Chapter 13 An Introduction to Cloning and Recombinant
Genetic Engineering Recombinant Cloning Transposons PCRs RFLPs Gel
Cloning Gene cloning replication of donor genes in
Cloning Nature has been cloning organisms for billions
Cloning CMC 1 Bach What is Cloning Reproductive
Cloning and genetic engineering by Ivo Frbort Cloning
Molecular Cloning Definitions q Cloning q Obtaining a