Cloning Vectors PDF

**CLONING VECTORS** - Small pieces of DNA taken from a virus or a plasmid that can be stably maintained in which foreign DNA fragments can be inserted for cloning purposes. PROPERTIES AND CONSTRUCTION OF VECTOR DNA MOL 1. AUTONOMOUS REPLICATION 2. SMALL SIZE - Chances of occurrenc...

**CLONING VECTORS** - Small pieces of DNA taken from a virus or a plasmid that can be stably maintained in which foreign DNA fragments can be inserted for cloning purposes. PROPERTIES AND CONSTRUCTION OF VECTOR DNA MOL 1. AUTONOMOUS REPLICATION 2. SMALL SIZE - Chances of occurrence of unique sites for RE increases - Efficiency of gene transfer is high 3. PRESENCE OF SELECTABLE MARKER GENES - Easy detection of recombinants - Antibiotic-resistant genes 4. MULTIPLE CLONING SITES - Insertion of a segment of DNA should bring a phenotypic change in the characteristic of a vector molecule - E.g., loss of gene expression 5. EASE OF PURIFICATION 6. NO EFFECT ON THE REPLICATIVE ABILITY OF THE VECTOR DUE TO THE INSERTION OF TARGET DNA 7. EASE OF REINTRODUCTION INTO HOST CELL WITH HIGH EFFICIENCY 8. BIOLOGICAL CONTAINMENT - Vectors should have no possibility of gene escape - Achieved by non-conjugative plasmid vectors 9. PRESENCE OF TWO DIFF ORIGIN OF REPLICATIONS - Shuttle vectors contain two different origins of replication 10. PRESENCE OF PROMOTORS AND RIBOSOME BINDING SITES. COMMON FEATURES OF VECTORS 1. ORI 2. Dominant selectable marker 3. Multiple cloning sites 4. High copy number CLONING SITE - Place where the vector DNA can be digested and same RE can insert desired DNA - Recombinant plasmids contain multiple cloning sites that have up to 20 restriction sites - Bacteria: single ORI - Eukaryotes: multiple ORI SELECTABLE MARKER - Gene that confers resistance to particular antibiotics or selective agent - Confers on the host cell an ability to survive and proliferate in a selective growth medium REPORTER GENE OR MARKER GENE - Used to facilitate screening of successful clones - Used in blue-white selection ADDITIONAL PROPERTIES OF VECTORS 1. Should be short, small 2. Compatible with the host cell 3. Incompatible with the other vector 4. High in copy number 5. Should be able to express itself using host machinery 6. Able to move under two systems. NATURAL PLASMIDS 1. pSC101 - Used for in vitro cloning of eukaryotic DNA - 9 Kbp in size - Low copy number - Advantage of single Eco RI at which DNA can be inserted - Has selectable markers for tetracycline resistance - Derived from conjugative plasmid R6-5 - Larger size - Stringent replicative control - Low copy number - Low insert capability - Plasmid of salmonella 2. Col E1 plasmid - Small, circular colicingenic plasmid - 6466 bp - Has ***cea*** gene for colicin production - ***Imm*** for immunity against colicin - ***Kil**l* for killer or lysis protein - ***Mob*** for mobilization - High copy number - Enriched by the addition of chloramphenicol 3. pSF2124 - produced by transfer of the ampicillin resistance gene - Has ability for colicin biosynthesis (Colicins are proteins produced by and toxic for some strains of Escherichia coli) - High copy number - Single sites for Bam HI and Eco RI - mobilizable plasmid MAIN CLASSES OF VECTORS 1. PLASMID: pUC 2. VIRAL: M13, Lambda PLASM*D PBR322* - Cloning vector that replicates in E. coli. - ORI allows independent replication - Single cleavage sites for various restriction enzyme - Tet and Amp resistance gene MECHANISM - Insertion of foreign DNA at the BamHI site - Tet resistance gene inactivated - Transformants carrying foreign DNA are amp resistance resistant but tetracycline sensitive TRANSFORMATION EFFICIENCY IS INVERSELY PROPORTIONAL TO THE SIZE AND ABOVE 10 Kbp IS VERY LOW. pUC SERIES - Next gen - Collects all restriction sites into MCS - Single, well-defined selection - Screening with X-gal PGEM3Z - Lambda based vector - Transduction and conjugation - Lytic cycle - Phage DNA properties - Introduces RNA expression VIRAL VECTORS 1. M13-BASED VECTOR - Ideal vector for sequencing - Excellent vector for phage display 2. Lambda based vector - Carry large amounts of DNA - Capable of in vitro packaging PHAGE DOUBLE-STRANDE DNA - Linear DNA - Non-essential gene for packaging of DNA MECHANISM 1. Attach to bacteria 2. Insertion of DNA 3. Specific regions 4. Take control of replication 5. Use bacterial DNA to reproduce their DNA 6. They burst and spread 7. COS SITE: 12bp sequence, cohesive sticking and form circular DNA M13 - Single stranded - Conjugation using pilli - SsDNA will form DsDNA then replication will happen - Does not kill the phage rather it slows down to 1/3