cloning steps using plasmid vector

Topics: Molecular biology, DNA, Plasmid Pages: 6 (1150 words) Published: December 2, 2014
UNIVERSITY OF DAR ES SAALAM

COLLAGE OF NATURAL AND APPLIED SCIENCE
DEPARTMENT OF MOLECURAL BIOLOGYG AND BIOTECHNOLOGY
GENE CLONING .
NAME: KANENDA. Ally. O
REG #: 2013-04-02841
DEGREE PROGRAM: MOLECURAL BIOLOGY AND BIOTECHNOLOGY
COURSE INSTRUCTOR: Dr. ROSE MASALU

Gene cloning:
Gene cloning is the process of inserting a gene of importance into a recipient organism, usually a bacterium, in order to replicate that gene many times and mass-produce a useful gene product. The recipient organism is called the host, and the material used to introduce the gene, usually a plasmid or bacteriophage, is called the vector. Ideal vectors have high rates of replication. Gene cloning is an important procedure in genetic engineering that allows us to produce a large amount of a useful gene product: for instance, insulin can be made for diabetic individuals by cloning genes from pancreatic cells.(Wikipedia 15/11/2014) Cloning vector:

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.( Genome Dictionary. Retrieved 2012-10-18.) The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector. There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial artificial chromosomes (BACs). Some DNA however cannot be stably maintained in E. coli, for example very large DNA fragment, and other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs). Plasmid is an autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the most commonly used. REASONS FOR USING PLASMID AS A VECTOR:

Most general plasmids may be used to clone DNA insert of up to 15 kb in size. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell, and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells. Some plasmids contain an M13 bacteriophage origin of replication and may be used to generate single-stranded DNA. These are called phagemid, and examples are the pBluescript series of cloning vector The pUC plasmid is a more advanced vector, whose structure allows direct visual selection of colonies containing vectors with donor DNA inserts.  The key element is a small part of the E.Coli beta-galactosidase gene.  Polylinker or multiple cloning site which contains many unique restriction target sites used for the gene insertion, inserted into the galactosidease gene.  The polylinker is in frame translationally with the galactosidase fragment and does not interfere with its translation.  Insertion of gene of interest to this lacZ gene used for their selection in X-gal medium depending upon the color development. i.e. active enzyme expression produce blue color whereas inactive enzyme produce the white color colonies. Selfreplicative nature and expression nature of the plasmids found to be the advantageous nature of plasmids.  Low copy number, small fragment insertion size and low transformation efficiencies found to be disadvantageous nature of plasmids.( STEPS IN DNA CLONING:

1. ISOLATION OF GENE OF INTEREST
The first step in cloning is to isolate the DNA from the organism that contains the desired gene. Then isolated DNA is purified and then cut with restriction enzyme which produces staggered cuts in specific sequences in the DNA, generating fragments with...
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