Clone: A group of identical cells or organisms
To clone a gene:
Insert the gene into a vector that can carry the into a host cell 2)
Ensure that it will replicate there
Insertion is carried out by cutting the vector and the DNA to be inserted with the same restriction endonuclease to ensure that both have the same "sticky ends" Vector for cloning the bacteria come from two major types:
Recombinant DNA Technology
It was developed in 1970
Obtain DNA from an organism
(2)ADD Restriction enzymes
A reproducible set of fragments
(4)Inset into replicating DNA molecules
This gives Recombinant DNA
Introduce this DNA in appropriate cells
Why clone a gene?
Let's say you have a Genome with GeneA, GeneB & GeneC
Your gene of interest is GeneA, You would
Select the gene of interest
Clone the gene, which could be used for
DNA sequence, Protein product expression
Prepare the gene product in large scale
Restriction digestion generates a reproducible set of DNA fragments(?) Restriction Enzymes:
Bacterial Enzymes that recognize a specific 4-8 base pair sequences in double
stranded DNA (2)
Cleave both DNA strands at the restriction sites.
Cut the DNA into a reproducible set of
fragments called restriction fragments.
Types of Nucleases
Endonucleases: Make a cut within a DNA molecule
Exonucleases: Digest DNA from the end Types of cuts By Endonocleases: (A)
Sticky Ends: Many restriction enzymes make staggered cuts in a DNA strand, leaving single-stranded overhangs or "Sticky Ends" Ex: EcoRI produces 4-base overhangs that protrude from 5' end of the fragments (B)
Blunt Ends(flush ends): Restriction Enzymes may cut in the middle of their Sequence leaving no overhangs. This type of ends are referred to as "Blunt Ends" Ex: AlvI cuts right in the middle of its sequence AGCT
Two types of gels are used for DNA...
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