DNA操作技术
1、Electrophoresis (Agarose):
separation of DNA molecules due to their negatively charged phosphate group.
用EB染DNA，在UV下看
2、Fluorescence in situ hybridization (FISH)
设计探针，显示荧光信号
3、southern blot：DNA杂交
DNA X DNA (in the gel)
High-throughput southern blot: microarray
4、northern blot：mRNA杂交
DNA X RNA (in the gel)
5、合成DNA
1)Primers (DNA sequencing, PCR)
2)Probes (Southern or Northern blots)
3)Site-directed mutagenesis定点突变
6、PCR (Polymerase Chain Reaction)
Denaturation变性—annealing退火—extension延伸
Thermus aquaticus, Taq polymerase, stable at 95 °C
Pyrococcus furiosus, Pfu polymerase, proofreading activity校对活性，高保真
Cloning genes or sequencing克隆测序
Comparative or phylogenetic analysis系统发育比较分析
Amplify very small amount of DNA扩增
Common tool for diagnostic microbiology疾病诊断
DNA fingerprinting DNA指纹分析
Reverse transcriptase PCR 逆转录PCR，表达分析以及真核基因克隆
Real-time qPCR实时定量PCR，连续检测扩增情况
·Accurately determine the amount of target DNA present in the original sample.准确确定DNA的原始量
·Access the abundance of an organism in a sample by quantifying a gene characteristic of that particular organism.
分子克隆Molecular Cloning
7、Conventional Cloning常规克隆
双酶切Restriction Enzymes: Commonly used type II restriction endonucleases 限制酶II cut the DNA within their recognition sequences (palindrome回文序列).
Most restriction enzymes are homodimeric proteins.同源二聚体蛋白
Modification enzymes: Methylation.
8、筛选
Insertional inactivation插入失活
Cells containing the vector with an insert of cloned DNA are white重组DNA成功就是白色的菌落, and cells containing the vector without cloned DNA are blue.空载体是蓝色的菌落（beta半乳糖苷酶将X-gal切成蓝色）
直接检测是否表达相关蛋白或含有特定序列
·Radiolabeled antibodies标记抗体 are used as detection reagents for a protein of interest.
·Nucleic acid probe核酸探针 labeled with radioactive phosphate 放射性磷酸盐or a fluorescent chemical group 荧光化学基团is used to detect nucleic acids containing specific sequences.
9、宿主选择
Escherichia coli | Bacillus subtilis | Saccharomyces cerevisiae |
原核生物 | 原核生物 | 真核生物 |
G- | G+ | 真核 |
有些致病 | 方便转化，不致病 | 不致病，安全性好 |
有周质空间，可能会形成包含体 | 没有周质空间，好培养 | 真核表达，mRNA和蛋白 |
遗传分析透彻 | 遗传分析不透彻，遗传上可能不稳定 | 质粒不够稳定，不会自主复制原核质粒 |
细菌表达外源蛋白
10、真核来源基因表达的障碍
Obstacles to the expression of genes from eukaryotic sources:
(1) Under control of a bacterial promoter启动子
(2) Remove introns内含子
(3) Codon usage密码子优化，适合菌体的tRNA库
(4) Bacteria cannot perform most host modifications after translation.后修饰
11、设计诱导
IPTG-诱导lac-T7 RNA聚合酶-目的基因诱导
12、通过mRNA逆转录克隆基因
真核mRNA:低丰度，多聚腺苷酸尾；含有与纤维素载体连接的聚(T)链的色谱柱，低盐缓冲液释放
过柱然后再RT-PCR
13、在载体上设置carrier protein，与目的蛋白组合成融合蛋白，方便蛋白从周质空间中分泌出去。然后再用酶切获得目的蛋白。
Molecular Methods for Mutagenesis诱变的分子方式
14、Site-directed mutagenesis定点突变，单链-双链
15、PCR引入突变：在引物上引入突变，只能在双链边上引入
16、加入中间两个引物，再用PCR引入突变。这样就能在双链中间突变。
17、大片段分子突变（基因敲除）
同源重组敲除（同源臂设计）
缺点是引入了抗性基因
Reporter Gene and Gene Fusions
18、Reporter systems: to assess the target promoter activity by transcriptional fusion; 转录报告基因
to determine locus of target protein by translational fusion.翻译报告基因
GFP对生物体无害，并且可传代
19、Transfection转染：将DNA导入哺乳动物细胞
Phagocytosis吞噬作用
Microinjection显微注射
Electroporation电转法
DNA gun基因枪
Genetically Modified Organisms（GMOs）基因工程生物
20、Mining Genomes挖掘基因组
Gene mining基因挖掘 is the process of identifying and isolating potentially useful genes 识别分离潜在有用基因from the environment从环境中 without the need to culture the organisms that contained them不需要培养包含它们的生物体.
Collect DNA samples from different environments；
Construct gene library建造基因文库；
Transform host cells and plate on selective medium转移到宿主细胞并用选择培养基；
Screen library for reactive colonies筛选文库
21、Pathway engineering组装代谢通路
assembling a new or improved biochemical pathway using genes from one or more organisms利用一种或多种生物的基因组装新的或改进的生化途径
比如Biofuel生物燃料、生物染料
22、Synthetic biology合成生物学
the use of genetic engineering to create novel biological systems out of available biological parts, often taken from several different organisms. 利用遗传工程从可用的生物部分创造新的生物系统，通常取自几种不同的生物体。
A. Library B. Assemble C. Installation D. Function
比如生产青蒿素