Recombinant DNA Technology & Library Construction Flashcards
What is recombinant DNA technology?
It is a set of techniques used for recombining genes from various sources in vitro which are transferred to a host cell where it may be expressed.
Requirements for recombinant DNA technology (6)
- gene/gene source
- vector
- enzymes (restriction endonuclease, ligase)
- transformation method
- host cell
- selection method
General Steps of recombinant DNA technology
- Restriction enzymes cut both the vector and the donor DNA at specific sites, creating overhangs
- the donor DNA is then inserted into the vector through a transformation method, producing recombinant vectors
- the recombinant vectors are inserted into a host cell to allow replication
- a method for selection is determined to detect successful transformation
enzymes isolated chiefly from prokaryotes that recognize specific sequences within the double-stranded DNA
restriction endonucleases
mechanism of RE
cuts at specific restriction site (short nucleotide sequence)
How do RE protect the bacteria?
It prevents foreign DNA from infecting/invading by degrading it
How do bacterial cells escape restriction digestion by their own REs?
the host DNA is modified by methylation (via methylase) which prevents recognition of the RE
Used RE type for recombinant DNA technology
type II
RE Type I
moves along the DNA before cutting the strand, and releasing a number of nucleotides where the cut is made
RE Type II
recognizes a specific target sequence and break the DNA within or close to the recognition site
RE Type III
intermediate of RE Types I and II
RE Type IV
targets methylated DNA
two possible resulting restriction fragments
- blunt ends
- overhangs
joining linear DNA
ligation
ligation enzyme
DNA ligase