Presentation Topic Cloning Vector and its Types Presented

Presentation Topic Cloning Vector and its Types Presented By Farah Mushtaq 117117 Faiza Saddique 117119

CLONING VECTORS A DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA

PLASMID Bacterial cells may contain extrachromosomal DNA called plasmids. Plasmids are usually represented by small, circular DNA. Some plasmids are present in multiple copies in the cell

PLASMID VECTORS Plasmid vectors are ≈1. 2– 3 kb and contain: replication origin (ORI) sequence a gene that permits selection, Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin. Exogenous DNA can be inserted into the bracketed region.

TWO MAIN FEATURES OF THE CLONING VECTOR ORIGIN OF REPLICATION SELECTABLE MARKER

ORIGIN OF REPLICATION Origin of replication is a DNA segment recognized by the cellular DNAreplication enzymes. Without replication origin, DNA cannot be replicated in the cell.

SELECTIVE MARKER Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker the plasmid becomes useful for the cell. Under the selective conditions, only cells that contain plasmids with selectable marker can survive Genes that confer resistance to various antibiotics are used. Genes that make cells resistant to ampicillin, neomycin, are used

MULTIPLE CLONING SITE Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other Restriction sites of the polylinker are not present anywhere else in the plasmid. Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

Gene to be cloned can be introduced into the cloning vector at one of the restriction sites present in the polylinker

TYPES OF CLONING VECTORS

CLONING VECTORS The vector is chosen according to the size and type of DNA to be cloned Different types of cloning vectors are used for different types of cloning experiments.

PLASMID VECTORS Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100 bp 10 kb).

ADVANTAGES OF PLASMIDS small size (easy to manipulate and isolate) circular (more stable) replication independent of host cell several copies may be present (facilitates replication) frequently have antibody resistance (detection easy)

DISADVANTAGES USING PLASMIDS range from 0 - 10 kb Standard methods of transformation Cannot accept large fragments Sizes are inefficient

BACTERIOPHAGE LAMBDA It infects bacteria Lambda phage vector High transformation efficiency, about 1000 times more than the plasmids vector Origin of replication Genes for head and tail protein Single-stranded producing cohesive ends

BACTERIOPHAGE LAMBDA

YEAST ARTIFICIAL CHROMOSOMES

Purpose: Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes Clone very large inserts of DNA: 100 kb - 10 Mb Features: YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome

Additional features: Often have a selection for an insert YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a selection marker for propogation of the YAC through bacteria. The YAC can use both yeast and bacteria as a host

RETROVIRAL VECTORS Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells. Retroviruses are RNA viruses. The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely. Retroviral vectors are widely used to study oncogenes and other human genes.

EXPRESSION VECTORS Allows a cloned segment of DNA to be translated into protein inside a bacterial or eukaryotic cell. Vectors will contain the ff: (a) in vivo promoter (b) Ampicillin selection (c) Sequencing primers

Produces large amounts of a specific protein Permits studies of the structure and function of proteins Can be useful when proteins are rare cellular components or difficult to isolate

REPORTER GENE VECTORS A gene that encodes a protein whose activity can be easily assayed in a cell in which it is not normally expressed These genes are linked to regulatory sequences whose function is being tested Changes in transcriptional activity from the regulatory sequences are detected by changes in the level of reporter gene expression

SHUTTLE VECTORS Shuttle vectors can replicate in two different organisms, e. g. bacteria and yeast, or mammalian cells and bacteria. They have the appropriate origins of replication. Hence one can clone a gene in bacteria, maybe modify it or mutate it in bacteria, and test its function by introducing it into yeast or animal cells

COSMID VECTOR Combine parts of lambda chromosomes with parts of plasmids An origin of replication(ori) A cos site (a sequence yield cohesive ends) An amplicine resistance gene (amp) Restriction sites for cloning Cosmids carry up to 50 kb of inserted DNA

APPLICATIONS A particular gene can be isolated and its nucleotide sequence determined Control sequence of DNA can be identified analyzed Protein/enzyme/RNA functions can be investigated Mutation can be identified, e. g. gene defects related to specific diseases Organisms can be engineered for specific purposes, e. g. insulin production

SUMMARY Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA Three features of all cloning vectors sequences that permit the propagation of itself in bacteria (or in yeast for YACs) a cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes a method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; uually accomplished by selectable markers for drug resistance

General Steps of Cloning with Any Vector prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends ligate the foreign DNA into the vector with the enzyme DNA ligase introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation select cells containing foreign DNA by screening for selectable markers (usually drug resistance)

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