Genetic Engineering
Scientific alteration of the structure of genetic material in a living organism. It involves the production and use of recombinant DNA and has been employed to synthesize insulin and other human proteins.
Bacteria are capable of modifying specific sequences within their genomes by methylation which prevents their own DNA from being recognized by the restriction enzymes encoded by their genomes. This process is termed modification and restriction (This system is called restriction-modification system). Infecting bacteriophage DNA is not modified and, hence, will be digested by the restriction endonucleases present in the bacterium
Recombinant DNA: is a form of artificial DNcombination or insertion of one or more DNA strands.
DNA cloning involves separating a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands of times. The result is a selective amplification of that particular gene or DNA segment.
I Principles
1. DNA cloning
Clone: A population of identical cells or DNA molecules descended from a single progenitor.
DNA cloning: refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it
Characteristics of restriction enzymes: 1. Cut DNA sequence specifically 2. Bacterial enzymes; hundreds are purified and available commercially 3. Restriction-modification system 4. Named (e.g., EcoR I) for bacterial genus, species, strain, and type 5. Recognize specific 4-8 bp sequences Sequences have symmetry (they are palindromes). After cutting the DNA, the cut ends are either blunt or staggered (overhangs) - cohesive ends facilitate cloning the DNA
To clone a piece of DNA, DNA is cut into fragments using restriction enzymes that recognize specific sequences of bases in DNA. The fragments are pasted into vectors that have been cut by the same restriction enzyme. Vectors (e.g., plasmids or viruses) are needed to transfer and maintain DNA in a host cell.
2. Tool enzymes
For gene cloning, they include: SCISSORS: restriction enzymes GLUE: DNA ligase
Restriction Endonucleases
Restriction endonuclease are enzymes that will recognize, bind to and hydrolyze specific nucleic acid sequences in double-stranded DNA
EcoR I
5' -G AATTC- 3' 3' -CTTAA G- 5'
Palindrome---A segment of double-stranded DNA in which the nucleotide sequence of one strand reads in reverse order to that of the complementary strand.