Recombinant DNA Technology Molecular Biology and Public Health ( 分子生物学与公共卫生 ) ------DNA/RNA manipulation 4 (DNA/RNA 操作 4) In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born-recombinant DNA technology. enetic engineering is the application of this technology to the manipulation of genes. 2.1 Correlative concepts Clone A clone is defined as a number of identical copies (molecules, cells or individuals) all derived from a common ancestor. Also named asexual multiplication. DNA Cloning DNA cloning involves separating a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands or even millions of times.
Recombinant DNA technology By artificial means, when a gene of one species is transferred to another living organism, it is called recombinant DNA technology. In common parlance, this is known as genetic engineering. Applications in enzymology restriction endonucleases DNA polymeraseⅠ reverse transcriptase DNA ligase Alkaline phosphatase terminal transferase Taq DNA polymerase 有些限制性内切酶虽然识别序列不完全相同, 但切割 DNA 后, 产生相同的粘性末端, 称为同尾酶 (isocaudarner) 这两个相同的粘性末端称为配伍未端 (compatible end) Bam HⅠ ATCC CCTA Bg lⅡ AATCT TCTAA CCTA + ATCC A + ATCT TCTA A ATCT CCTAA The cohesive ends generated from the two enzymes pair with each other perfectly. But the hybrid ligation product cannot be cut by either enzyme.
Vector The term vector here refers to some DNA molecules that can carry a DNA fragment into a host cell for replication. Including: plasmids, Bacteriophages DNA, virus DNA eneral features of a Vector 1. Contains an origin of replication and can autonomously replicate its DNA independent of host s genome replication. 2. Can be easily isolated from the host cell. Most are circular, some are linear (e.g. YAC vector). 3. Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those not containing the vector. 4. Contains a multiple cloning site (MCS) that can be cut by restriction enzymes for DNA manipulation. 10 Cloning vectors ( 克隆载体 ): allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector (circular): plasmids ( 质粒 ) bacteriophages ( and M13) ( 噬菌体 ) plasmid-bacteriophage hybrids (cosmids) ( 考斯质粒 - 质粒和噬菌体杂和体 ). Yeast cloning vector: yeast artificial chromosomes (YACs, 酵母人工染色体 ) (Linear) Vector (and host) Vectors used in molecular cloning Insert Characteristics size range Plasmid Small circular DNA <5-10 kb (bacteria, yeast) Bacteriophage λ Linear viral DNA up to ~20 kb (bacteria) Cosmid Hybrid of plasmid up to ~50 kb (bacteria) and phage Yeast artificial DNA containing yeast ~200 to chromosome (YAC) centromere, telomere, ~1000 kb (yeast) and origin of replication 11
Plasmid Plasmid Plasmids are small, circular molecules of DNA that exist outside the main bacterial chromosome and carry their own genes for specialized functions. Plasmids as cloning vectors The idea arose that foreign DNA sequences could be inserted into artificial plasmids and that these foreign sequences would be carried into E. coli and propagated as part of the plasmid. These plasmids could serve as cloning vectors to carry and amplify genes of interest. Plasmids useful as cloning vectors possess three common features: A replicator: is an origin of replication, or ori. A selectable marker: is typically a gene conferring resistance to an antibiotic. Only cells containing the cloning vector will grow in the presence of the antibiotic. Therefore, growth on antibiotic-containing media selects for plasmid-containing cells. A cloning site: a sequence of nucleotides representing one or more restriction endonuclease cleavage sites. Cloning sites are located where the insertion of foreign DNA neither disrupts the plasmid s ability to replicate nor inactivates essential genes for plasmid maintenance and selectable marker resistance.
4363bp How can a DNA sequence be cloned? Nuclease cleavage at a restriction site opens, or linearizes, the circular plasmid so that a foreign DNA fragment can be inserted. The ends of this linearized plasmid are joined to the ends of the fragment so that the circle is closed again, creating a recombinant plasmid. Cleavage at single specific site by a restriction endonuclease ori Join free ends to ends of foreign DNA Foreign DNA
Foreign DNA fragment Cleavage with Sal I helps avoid purification of large fragment, why? Recombinant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus inserted foreign DNA elements (called inserts). Such hybrid molecules are also called chimeric constructs or chimeric plasmids. The presence of foreign DNA sequences does not adversely affect replication of the plasmid, so the chimeric plasmid can be propagated in bacteria just like the original plasmid. Limitation DNA sequences that are difficult to clone include inverted repeats, origins of replication, centromeres, and telomeres. The only practical limitation is the size of the foreign DNA segment: most plasmids with inserts larger than 10kbp are not replicated efficiently.
Bacteriophage λ as cloning vector The genome of bacteriophage λ is 48.5kbp linear DNA molecule that is packaged into the bacteriophage shell. The middle one-third of its genome is not essential to phage infection, so λ phage DNA has been engineered to accommodate the insertion of foreign DNA molecules, upto 16kbp, to package the chimeric DNA into phage heads which, when assembled with phage tails, form infective phage particles. Bacteria infected with recombinant phage produce large numbers of phage progeny before they lyse, and large amounts of recombinant DNA can be easily purified from the lysate. Phage phage DNA: gt phages: Insertion type vector EMBL phages: replacement type vector M13 phage: M13mp and puc
EMBL phages Cosmids The DNA incorporated into phage heads by bacteriophage λ packaging system must satisfy only a few criteria. It must possess a 14bp sequence known as cos (which stands for cohesive end site) at each of its end, and these cos sequences must be separated by no fewer than 36kbp and no more than 51kbp of DNA. Any DNA satisfying these minimal requirements will be packaged and assembled into an infective phage particle. Cloning features, such as an ori, selectable markers, and polylinker, are joined to the cos sequence so that the cloned DNA can be propagated and selected in host cells. These features have been achieved by placing cos sequences on either site of DNA inserts about 40kbp in size. Because cosmids lack essential phage genes, they reproduce in host bacteria as plasmids.
Shuttle vectors Shuttle vectors are plasmids that can propagating and transferring genes between two different organisms, one of which is typically a prokaryote (e.g. E. coli.) and the other a eukaryote (for example, yeast). Shuttle vectors must have unique origins of replication for each host type as well as different markers for selection of transformed host cells harboring the vector. Shuttle vectors have the advantage that eukaryotic genes can be cloned in bacterial hosts, yet the expression of these genes can be analyzed in appropriate eukaryotic backgrounds. 2 Recombinant DNA Technology
Process of cloning Isolation of target gene Selection and construction of vectors Ligation of target DNA and vector Transformation of target gene-vector ligation mixture into recipient cells Screening for recombinant plasmids Amplification/Expression of cloned target genes 1. Restriction digestion of your insert and vector using the same enzyme. 2. Use ligase to join your insert and vector together. 3. Transform the ligation products into E. coli. competent cells. 4. row the cells on a plate containing tetracycline ( 四环素 ). 38 Process of DNA cloning 2.1 Isolation of target gene 1. Chemical synthesis Only for simple polypeptide chain whose primary structure is clear. 2. Obtaining from genomic DNA library 3. Obtaining from cdna library 4. polymerase chain reaction (PCR)
The genomic DNA library is a collection of the comprehensive DNA fragments representing the entire genome of a species. Different Insert fragments 42 mrna The cdna library represents the population of cdna mrnas, it only contains the exons dscdna of protein s vector structural genes. recombinate DNA Reverse transcripase replication E. coli Preparation of cdna library recombinate DNA in E. coli
同一限制酶切位点连接 2.2. Selection and construction of vectors A few commonly used vectors: plasmid phage cosmid yeast artificial chromosome (YAC) 2.3 Ligation of target DNA and vectors 1. Ligation of sticky end ATCC CCTA ATCC CCTA ATCC CCTA ATCC CCTA ATCC CCTA DNA ligase ATCC CCTA CCTA Bam H CCTA CCTA ATCC CCTA ATCC CCTA ATCC Bam H DNA Bam H ATCC + ATCC T4 DNA 15ºC ATCC ATCC CCTA CCTA ATCC ATCC CCTA CCTA Eco R AATTC CTTAA AATTC 不同限制酶切位点的连接 Bg l AATCT TCTAA EcoR + Bg l A TCTA AATTC CTTAA AATCT TCTA A AATTC ATCT A T4 DNA 15ºC AATTC CTTAA AATCT TCTA A + Eco R + Bg l AATTC A TCTA 配伍末端的连接情况和同一限制酶切位点连接相似
Dephosphorylate the vector using alkaline phosphate can prevent religation of vector molecules Recombinant DNA molecules X if the vector is in the phosphorylated state 2. Ligation of blunt ends Foreign DNA Vector 3. The addition of a homopolymer tail Restriction enzyme Restriction enzyme recombinant T4 DNA ligase 15ºC Vector links itself Foreign DNA Links itself
4. Artificial linker Artificial linker Adding a sequence of DNA fragment, which contains the cleavage site for a restriction endonuclease. 2.4 Introduction of recombinant DNA into recipient cells Introduction: transformation transfection infection
Recipient cells Safe host bacteria Endonuclease and recombinase deficiency Competent cells: Competent is a physiological state that allows DNA uptake by the cell. 2.5 Screening for recombinant Screen of antibiotic resistance markers Marker rescue (Insertion inactivation) In situ hybridization and autoradiography direct selection Antibiotic resistance genes The procedure to form recombinant DNA direct selection
Screen of antibiotic resistance markers ampicillin ampicillin Blue white screening ---Allow the discrimination of recombinant plasmid from the religated ones Marker rescue 64 Amp r ori puc18 (3 kb) Lac promoter MCS (Multiple cloning sites, 多克隆位点 ) lacz Insertion of a DNA fragment interrupts the ORF of lacz gene, resulting in non-functional gene product that can not digest its substrate x-gal.
lacz encode enzyme b-galactosidase lac promoter IPT X-gal Blue product (substrate of the enzyme) Recreated vector: blue transformants Recombinant plasmid: containing inserted DNA: white transformants Recreated vector (no insert) During the experiment, IPT is added to the selective growth medium. Insertion of the target DNA fragment into the MCS will prevent the formation of the blue colony, and white colony is formed instead. Recombinant plasmid (contain insert) 66 In situ hybridization and autoradiography α 互补的检测
Colony hybridization-southern blot Transfer to nitrocellulose or nylon membrane Analysis of DNA clones Analysis of a clone Keep master plate Select positive from master plate Probe with 32 p-labled DNA complementary to gene of interest Expose to film Denature DNA(NaOH) Bake onto membrane 1.Restriction mapping: digestion of the plasmid prepared from a clone with restriction enzymes to investigate if the interested DNA is inserted into the recombinant plasmid. 2.Sequencing the cloned DNA to see if the inserted DNA maintains the correct sequence. Screening by plaque hybridization 70 1 Kb + ladder 2 Positive clones digested with different restriction enzymes Empty vector Sequencing Restriction mapping 72
2.6 Expression of the cloned gene An expression vector is similar to cloning vectors, but with a major difference: the expression vector must contain a promoter so that proteins can be expressed. ene expression Expression of a gene from a transformed/transfected plasmid 1. Transformation ( 转化 ) : introduction of plasmids into bacteria. 2. Transfection ( 转染 ): introduction of plasmids and other exogenous nucleic acids into eukaryotes such as mammalian cells. 74 Expression vectors: Allowing the exogenous DNA to be stored and expressed in an organism. --E. coli expression vector --Yeast expression vector --Mammalian expression vector Features: In addition to the origin of replication, selective marker, multiple cloning site, expression vector must contain a promoter and terminator for transcription. The inserted gene has to have a start codon and a T7 promoter ori stop codon for translation. 75 76 Amp r T7 expression vector RBS Start codon MCS Transcription terminator E. coli expression vector
Expression vector Fusion proteins ene cloning and expression provides a very powerful way to obtain a large amount of the target protein fused with an enzyme, fluorescence protein or a tag for identification ( 鉴定 ) or purification ( 纯化 ). 78 Examples Lac fusions (Enzyme) : fuse your target gene with the LacZ coding sequence. His-tag fusions (Tag) : A sequence encodes His-tag was inserted at the N- or C- termini of the target ORF, allowing the fusion protein to be purified by binding to Ni 2+ column. FP fusions (Fluorescence protein): insert your targeted gene at the N- or C- termini of FP (green fluorescence protein, 绿色荧光蛋白 ), and your fusion protein will give you Home work 1. When a vector and a candidate gene were cut by the same two restriction enzymes, E and H, to generate two different cohesive ends, name at least 3 possible circular ligation products that can be formed in the ligation mixture of the candidate and the vector. 2. What features an expression vector must have? green fluorescence signal. 79 80