基因克隆原理

b. Baterial initiation of protein synthesis begins near a Shine-Dalgarno
sequence (SD sequence)
mRNA --AGGAGGU—XXXX---AUG
c. Eukaryotic initiation of protein synthesis occurs at the 5’ end and internal
博士班分子生物学实验技术课
引言
分子生物技术是一门激动人心的、充满革命性 的学科，它始于研究者能将某些特定的遗传信 息单位从一个生物体转入另一个生物体内。
1980年10月14日，纽约证券交易所仅开盘20 分钟，Genentech生物技术公司的股票就从每 股35美元飙升到89美元。这也许是第一次一个 重大的技术革命被股票交易所的叮当铃声所证 实。
d. Many DNA Molecules Are Circular
e. Local Unwinding of DNA Induces Supercoiling
f. RNA Molecules Exhibit Varied Conformations and Functions
2. General Rules for Synthesis of Biopolymers
From 《Molecular Cell Biology》 4th ed
请大家看一段Plasmid Cloning 的Movie
Plasmid Cloning
Definition of cloning
• CLONE
– a population of identical cells or DNA molecules descended from a a single progenitor. Also viruses or organisms that are genetically identical and descended from a single progenitor
Bill Gates
关键生物化学知识的复习
1. Structure of Nucleic Acid 2. Synthesis of Biopolymers 3. Nucleic Acid Synthesis 4. Three roles of RNA in Protein Synthesis 5. Stepwise Formation of Proteins on
b. Native DNA Is a Double Helix of Complementary Chain
Mirror images; A DNA, B DNA, Z DNA, Triple-helical DNA; Modification of B DNA
c. DNA can Undergo Reversible Strand Separation
d. Ribosomes are protein-synthesizing machines
5. Stepwise Formation of Proteins on Ribosome
a. The AUG start codon is recognized by methionyl-tRNAiMet, deffering from for tRNAMet elongaton
Dr.James Watson
“[If I were not in computers] I’d be working in biotechnology. I expect to see breathtaking advances in medicine over the next two decades, and biotechnology researchers and companies will be at the center of the progress.”
f. Eukaryotic primary RNA transcripts are processed to form functional mRNAs
4. Three roles of RNA in Protein Synthesis
a. Messenger RNA carriers information from DNA in a three-letter genetic code 43=64 codons; start codon: AUG; Stop codon: UAA, UGA and UAG. reading frame 20 amino acids: A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y Experiments with synthetic mRNAs and trinucleotides broke the genetic code --------a great triumph in biochemistry
质粒载体的命名，首字母用小写p表示，后面是 一些描述性的缩写，如:pBR322的字母代表研究 者F.Bolivar和R.Rodriguez, 而322是与研究者设 计相关的数字编码。
质粒载体举例
(1) the replicon rep responsible for the replication of plasmid; (2) rop gene coding for the Rop protein, which promotes conversion of the unstable
c. Each polypeptide and polynucleotide chain has a specific starting point, and growth proceeds in one direction to a fixed terminus.
d. The primary synthetic product is often modified.
f. Simultaneous translation by multiple ribosomes and their rapid recycling increase the efficiency of protein thesis
基因克隆原理
特异DNA序列的一般研究方法
General procedure for cloning a DNA fragment in a plasmid vector
d. Replication of duplex DNA requires assembly of many proteins at a growing folk
e. Organization of genes in DNA differs in prokaryotes and eukaryotes
site (Kozak sequences) in mRNA.
mRNA --ACCAUGG----AUG
d. During chain elongation each incoming aminoacyl-tRNA moves through three ribosomal sites
e. Protein synthesis is terminated by release factors when a stop codon is reached.
Cloning vectors
• Plasmids • lambda (l) vectors • cosmids • BACs and YACs
质粒是细菌染色体外小型双链环状DNA复制子,对细菌的 某些代谢活动和抗药性表型具有一定的作用。
质粒载体是在天然质粒的基础上人工改造拼接而成。
作为载体的质粒一般具备以下特点： 1）分子相对较小； 2）具有松弛型复制子； 3）在复制子之外适当位置， 存在多克隆位点； 4）具有筛选标记。
RNA I - RNA II complex to a stable complex and serves to decrease copy number ; (3) bla gene, coding for beta-lactamase that confers resistance to ampicillin; (4) tet gene, encoding tetracycline resistance protein.
3. Nucleic Acid Synthesis
a. Both DNA and RNA chain are produced by copying of template DNA strand
b. Nucleic Acid strands grow in the 5’ 3’
c. RNA polymerases can initiate strand growth but DNA polymerases cannot RNA or DNA primer
• VECTOR
– the vehicle that transports the DNA molecule in the host cell
• CLONING VECTOR
– An autonomously replicating genetic element used to carry a cDNA or fragment of genomic DNA into a host cell for the purpose of gene cloning. Commonly used vectors are bacterial plasmids and modified bacteriophage genomes.
• DNA CLONING
– Recombinant DNA technique in which specific cDNAs or fragments of genomic DNA are inserted into a cloning vector, which then is incorporated into cultured host cells and maintained during growth of the host cells; also called gene cloning
The Farm 2000 Alexis Rockman oil and acrylic on wood panel, 96 x 120 in.
“We used to think our fate was in our stars. Now we know……our fate is in our genes.”
Ribosome
1. Structure of Nucleic Acid
a. Polymerization of Nucleotides Forms Nucleic Acids
Pentose + Base Nucleoside + Phosphate Nucleotide Nucleic Acid
DNA RNA Protein


Leabharlann Baidu
a. Protein and nucleic acids are made up of a limited number of different
monomeric building blocks.
b. The monomers are added one at a time.
b. The folded structure of tRNA its decoding functions; Nonstandard base pairing often occurs between codons and anticodons (20 a.a. < 30-40 tRNAs <61 codons); 20 aminoacyl-tRNA synthetases activate amino acids by linking them to tRNAs;