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Recombinant DNA and Genetic Engineering (Restriction - modification…
Recombinant DNA and Genetic Engineering
Recombinant DNA
Segments of DNA from two or more sources linked together
They are combined in vitro into the same DNA molecule
Usually DNA sequences that would not normally occur together
Gene cloning: Involves using a host cell to make multiple copies of a gene.
Foreign DNA Is inserted into a plasmid and the recombinant plasmid is inserted into a bacterial cell
Reproduction in the cell results in cloning the plasmid
Recombinanat DNA tech based on:
Endonucleases - Enzymes which cut DNA into fragments (DNA fragment = the insert)
Commonly called 'Restriction Enzymes'
Use of Vectors
Bacteriophages: Viruses that invade bacteria and replicate their own DNA independently of the bacterial chromosomal DNA
Exonuclease cuts nucleotides from end of a DNA molecule
Endonuclease cleaves internal phospodiester bonds
3 types of restriction endonucleases
Type 1- Cut at random
Type 2 cut at a defined position - only type used in DNA analysis
Type 3 -cut at 25bp from recognition site and requires ATP
Type 2 restriction enzyme
each enzyme has a specific sequence at which it cuts a DNA molecule - This is the recognition sequence. A particular enzyme will only cut at that site and nowhere else
Restriction - modification systems
Naturally occur in bacteria in combination with modification enzymes : DNA-Methyl transferases
protect bacterium own DNA cleavage
Modification enzymes recognize the same DNA sequence as the restriction enzyme that they acoompany
They do no cleave the sequence, they methylate to one of the bases in each of the DNA strands
Methyl groups prevent the restriction enzyme from cutting the DNA
DNA Ligases
A naturally occurring enzyme which in the cell repairs single-stranded breaks in double stranded DNA
Ligation of sticky-ended molecules - sticky ends increase efficiency of ligation
Bacteria are useful hosts:
Easy to manipulate
Rapid growth and cell divison
Have genetic markers so easy to select or screen for insertion of DNA of interest
E.coli / gram negative bacterium an example
Bacteria disadvantages
Lack splicing machinery to excise introns
Protein modifications are not done as they would in a eukaroytic cell