Section G-I Gene Manipulation G1 DNA cloning: an overview G2 Preparation of plasmid DNA G3 Restriction enzymes Section G: Gene manipulation Yang Xu, College of Life Sciences

G1 DNA Cloning: An Overview Sub-cloning DNA libraries Screening libraries Vectors Plasmids Hosts Analysis of clone Section G: Gene manipulation Yang Xu, College of Life Sciences

DNA cloning Definition: It is the making process to place the target gene (the gene of interest) in a vector (an autonomously replicating piece of DNA), forming recombinant DNA, which then is placed into another host species. E S target gene Host transformant Ampr vector Ori Section G: Gene manipulation Yang Xu, College of Life Sciences

DNA cloning: Basic Process Example: plasmid (vector) and E. coli (host) (1) Preparation of plasmid DNA containing the cloned target gene. (2) Digestion of the plasmid with restriction endonucleases. (3) Separation of the fragments by agarose gel electrophoresis. (4) Purification of the desired target fragment. (5) Ligation of the fragments, to form a new recombinant molecule. (6) Transformation of the ligated plasmid into an E. coli strain. (7) Selection of transformed bacteria (see Topic G4). (8) Analysis of recombinant plasmids (see Topic G4). Section G: Gene manipulation Yang Xu, College of Life Sciences

Subcloning Definition: It is the process to transfer of a fragment of cloned DNA from one vector to another. Experimental steps: 1. Preparation: of the plasmid 2. Digestion of the plasmid 3. Separation of the fragments 4. Purification of target fragment 5. Ligation of the fragments 6. Transformation 7. Selection of transformed bacteria. 8. Analysis of plasmids. ① ② ③ + - ⑦ ⑥ ⑤ ⑧ ④ Analysis Section G: Gene manipulation Yang Xu, College of Life Sciences

DNA libraries Definition: DNA libraries are sets of DNA clones (vectors/hosts), each of which has been derived from the insert of a different fragment into a vector followed by propagation in the host. Classification and features: 1. Genomic libraries: They are derived from random fragments of DNA from the genomes of species by shotgun approach; The approach may be an inefficient of finding a gene, especially in eukaryotic genomes, where much of the DNA is noncoding. Section G: Gene manipulation Yang Xu, College of Life Sciences

DNA libraries 2. cDNA libraries: (ProteinCellmRNAcDNAVectorHost) They are derived from the mRNA by reverse transcription and are then inserted into a vector; cDNA libraries are efficient for finding and cloning a gene, but only the coding region, not the surrounding genomic sequences. Section G: Gene manipulation Yang Xu, College of Life Sciences

Screening libraries Definition: It is the process to use a DNA probe to find the clone that contains the gene interested. DNA probe: It is a radioactively labeled short DNA sequence which is partially complementary to a region of the target gene sequence, therefor, the target gene or clone can be detected by its hybridization. Making of the probes: The probe sequence might be an oligo-nucleotide derived from the sequence of the protein product of the gene; From a related gene from another species. An increasingly important method for the generation of probes is PCR Section G: Gene manipulation Yang Xu, College of Life Sciences

Vectors-I: Features of vectors The features of vectors: Vectors must normally be capable of being replicated and isolated independently of the host's genome; Vectors also have a selectable marker, a gene which allows host cells conferring resistance to a toxin. There are some vectors, for example phage  (see Topic H2), which can incorporate DNA into the host genome for longer term expression of cloned genes. B tetA ampr Ori pBR322 Section G: Gene manipulation Yang Xu, College of Life Sciences

Vectors-II: Types of vectors The common vectors: 1. Plasmids: Circular plasmid of E. coli: used in E. coli (host); Yeast episomal plasmids: used in yeast; Agrobacterium tumefaciens Ti plasmid: in plant. 2. Bacteriophages (viruses infecting bacteria; see Topic R2): Phage : also been used in E. coli, for cloning larger fragments Phage M13: used to clone ssDNA used in E. coli. 3. Cosmids (plasmid-bacteriophage hybrids): (see Topic H3). 4. Artificial chromosomes: for cloning huge fragments from humans. BAC: Bacterial artificial chromosomes (in E. coli); YAC: Yeast artificial chromosomes (in Yeast). 5. Virus: for other eukaryotic cells in culture SV40; Retroviruses. (see Topic H4). Section G: Gene manipulation Yang Xu, College of Life Sciences

Plasmids The first cloning vectors to be used, in the mid 1970s, were natural plasmids originally from E. coli. Structure and features: Plasmids are  small in size, from 2 to around 200 kb  extrachromosomal circular molecules  which exist in multiple copies (up to a few hundreds)  within the host E. coli cells.  They contain an origin of replication (ori), which enables them to be replicated independently. Section G: Gene manipulation Yang Xu, College of Life Sciences

Plasmids Resistance gene: They usually carry a few genes, one of which may confer resistance to antibacterial substances: 1. The most widely known resistance gene is ampr gene, which encodes the enzyme -lactamase, that degrades penicillin antibiotics such as ampicillin. 2. Another is the tetA gene, which encodes a transmembrane pump able to remove the antibiotic tetracycline from the cell. Section G: Gene manipulation Yang Xu, College of Life Sciences

Hosts The common hosts: E. coli: The initial isolation and analysis of DNA fragments is almost always carried out using the E. coli as the host (for circular plasmid, phage , phage M13, cosmid, BAC); Yeast: It is being used to manipulate very large fragments of the human genome (for episomal plasmid, YAC). Other cells: Agrobacterium tumefaciens (for Ti plamid); Insect cells (for baculovirus); Eukaryotic cells (for SV40, retroviruses, shuttle vectors). Section G: Gene manipulation Yang Xu, College of Life Sciences

Analysis of clone Once a clone containing a target gene is identified, the structure of the cloned fragment may be investigated: 1. further using restriction mapping, the analysis of the fragmentation of the DNA with restriction enzymes (see Topic J1) and by agarose gel electrophoresis using marker of known sizes (see Topic G3), 2. or ultimately by the sequencing of the entire fragment (see Topic J2). 3. The sequence can then be analyzed by comparison with other known sequences from data bases, and the complete sequence of the protein product determined (see Topic J2). Section G: Gene manipulation Yang Xu, College of Life Sciences

G2 Preparation of Plasmid DNA Bacterial culture Alkaline lyses Phenol extraction Ethanol precipitation Cesium chloride gradient Section G: Gene manipulation Yang Xu, College of Life Sciences

Plasmid mini-preparation Harvest cells By centrifugation Cells 1. Resuspend cell pellet; 2. Add lysozym 3. Add detergent/NaOH; 4.Neutrize with Plasmid RNA Protein Protein, chromosomal DNA and membranes Denatured protein Phenol-chloroform Chromosomal/ inear DNA (plasmid + RNA) Aqueous Pure plasmid Take aqueous 1. Ethanol precipitate 2. RNase 1. Collect supercoiled plasmid band 2. Ethanol precipitate Extraction: CsCl gradient Plasmid mini-preparation KOAc Section G: Gene manipulation Yang Xu, College of Life Sciences

G3 Restriction Enzymes and Electrophoresis Digestion, separation and purification Restriction endonucleases Restriction sequences Cohesive ends Restriction digests Agarose gel electrophoresis Section G: Gene manipulation Yang Xu, College of Life Sciences

Digestion: Restriction endonucleases Function: Restriction-modification systems occur in many bacterial species, and constitute a defense mechanism against the foreign DNA into the cell. Structure: Restriction-modification system consist of two parts： 1. The first part is a restriction endonuclease, which recognizes a short, symmetrical DNA sequence (Fig. 1), and hydrolyzes the DNA backbone in each strand at a specific site. 2. The second part is a methylase, which adds a methyl group to a C or A base within the same recognition sequences. This modification protects the host DNA against the endonuclease. Section G: Gene manipulation Yang Xu, College of Life Sciences

Digestion: Restriction sequences Definition: It is a short palindromic sequences, at which restriction enzymes cleave DNA symmetrically in both strands. Acting Steps: EcoRI as an example. Recognizing: The restriction endonucleases EcoR I acts as a dimer, will only recognize a 6 bp palindromic sequence. Cutting: The product of the cutting reaction is two restriction fragments, each with a 5'-end with a phosphate group and a 3'-end with a free hydroxyl group. EcoR I Recognizing 5’-GAATTC-3’ 3’-CTTAAG-5’ Cutting 5’-G-OH P-AATTC-3’ 3’-CTTAA-P HO-G-5’ annealing 5’-GAATTC-3’ 3’-CTTAAG-5’ Section G: Gene manipulation Yang Xu, College of Life Sciences

Digestion: Cohesive ends Definition: Some of the products of restriction enzyme digestion have protruding ends, and these ends are known as cohesive, or 'sticky' ends. Features: Those products of restriction enzyme digestion with protruding ends have a further property: They can bind to any other end with the same overhanging sequence, by base pairing (annealing) of the single-stranded tails. For example, any fragment formed by an EcoR I cut can anneal to any other fragment formed in the same way, and may subsequently be joined covalently by ligation. In fact, in some cases, DNA ends formed by enzymes with different recognition sequences may be compatible Section G: Gene manipulation Yang Xu, College of Life Sciences

Digestion: Restriction digests-I Application: Digestion of plasmid or genomic DNA is carried out with restriction enzymes for analytical or cloning preparation purposes. Examples: The digestion of a sample plasmid with two different restriction enzymes, Bam HI and EcoR I. E B Plasmid with gene X E B EcoRI BamHI B Section G: Gene manipulation Yang Xu, College of Life Sciences

Digestion: Restriction digests-II Reaction system: All restriction enzymes require Mg2+ (magnesium) usually at a concentration of up to 10 mM; but different enzymes require different :  pHs,  NaCl concentrations or  other solution constituents. Reaction process: The DNA is  incubated at the  optimum temperature (37C) with  the enzyme and  the appropriate buffer, in a volume of perhaps 20 l.  A dye mixture is then added to solution, and  the sample is loaded on to an agarose gel. Section G: Gene manipulation Yang Xu, College of Life Sciences

That’s all for Section G-I Section G: Gene manipulation Yang Xu, College of Life Sciences