RECOMBINANT DNA TECHNOLOGY Flashcards
TECHNOLOGY THAT ENCOMPASSES A NUMBER O F EXPERIMENTAL PROTOCOLS
LEADING T O TRANSFER O F DNA FROM ONE ORGANISM T O ANOTHER
Recombinant DNA TECHNOLOGY
Molecular scissors that cut DNA into fragments at specific sites in their sequence.
Restriction endonucleases
EcoRI
“Eco” word from?
1st letter of genus +
1st and 2nd letter
of species
EcoRI
“R”from ?
Strain of host bacteria
EcoRI
“I”from ?
Order of discovery
CLASSIFICATION OF RESTRICTION ENZYMES
Cut DNA at random far from their recognition sequences.
Type 1
Cut DNA at defined positions close to or within their recognition sequences.
Type 2
Cut modified (e.g. methylated) DNA.
Type 4
Cut DNA outside of two recognition sequences in opposite orientations.
Type 3
MECHANISM OF RESTRICTION ENZYMES
1.
2.
3.
- SCAN - Look fo a particular pattern of
nucleotides:ENZYME’S RECOGNITION
SEQUENCE - BIND- The enzyme will bind the DNA
molecule. - CUT- The enzyme cuts in each of the two
sugarphosphate backbones of the
double helix
Alul and Haelll produce _____ ends.
blunt
DNA sequence that produce blunt ends.
Alul and Haelll
BamHl, Hindlll and EcoRi produce ____ ends
sticky
Made by fusing DNA-binding domain to a nuclease domain.
Ex. CRISPR technolog
Artificial Restriction enzymes
DNA sequence that produce “ sticky” ends
BamHl, Hindlll, and EcoRi
Who won nobel prize in Chem 2020, “for the development of a method for genome editing.
Emmanuelle Charpentier
Jennifer A. Doudna
A DNA MOLECULE IN WHICH FOREIGN DNA
MOLECULE IS INSERTED AND WHICH IS FURTHER CAPABLE OF REPLICATING WITHIN HOST CELL TO PRODUCE MULTIPLE CLONES OF THE RECOMBINANT DNA.
CLONING VECTOR
MAIN FEATURES OF CLONING VECTORS
- ORIGIN OF REPLICATION
- MULTIPLE CLONING SITES
- SELECTABLE MARKER
DNA segment with several RE sites located next to each other.
MULTIPLE CLONING SITE
Allows selection of host cells with the recombinant vector.
Selectable marker
CLONING STRATEGIES
_________ involves the ligation of short, staggered ends that can be rejoined by DNA ligase to regenerate original recognition
sequence.
Cohesive End Cloning
CLONING STRATEGIES
_____ involves ligating dsDNA
into a plasmid where both the insert and
linearized plasmid have no overhanging bases
at their termini.
Blunt End Cloning
10-100x less efficient than cohesive-end
cloning. Desired insert does not require any restriction sites.
Blunt End Cloning
CLONING STRATEGIES
Modified Blunt End Cloning may use…
- Linkers
- Adaptors
- Homopolymer tails
final step in the construction of a recombinant
DNA molecule where the vector molecule and
the DNA to be cloned are joined together by
DNA ligase.
LIGATION
Two families of dna ligases
1.ATP-dependent Ligase
2. NAD+-dependent ligases (LigA)
All known eukaryal cellular DNA ligases; also in archaea.
ATP-dependent Ligase
distinctive and structurally homogeneous clade of enzymes found in all bacteria.
NAD+-dependent ligases (LigA)
is the process by which an organism
(competent) acquires exogenous DNA.
TRANSFORMATION
It is a Direct Methods of transformation,
- CaCl2
- TSS Buffer with PEG, DMSO, and MgCl2
- Usually followed by heat-shock transformation.
Chemical Competency
It is a Direct Methods of transformation,
_____ significantly increases the electrical
conductivity of cell membrane for
subsequent increase in its permeability.
Electrocompetency
It is a Direct Methods of transformation,
the use of a glass micropipette to inject a liquid
substance at a microscopic or borderline
macroscopic level.
Microinjection
It is a Direct Methods of transformation,
- Transforming yeast cells by limited physical
disruption in the presence of DNA - This method is less efficient but considerably
more convenient
Agitation by glass beads
4 INDirect Methods of transformation.
Transduction
Agrobacterium Mediated
Protoplast Fusion
Conjugation
2 METHODS OF SELECTION
1.Use of markers
2.Use of reporter genes
ANTIBIOTICS
-Positive selection
* Ex. Ampicillin
under what methods if selection?
METHODS OF SELECTION: 1. USE OF MARKERS
Negative selection
BLUE-WHITE SCREENING
The genes encode protein products that can
be observed using certain biochemical
assays.
METHODS OF SELECTION: 2. Reporter genes
Examples of Reporter genes. The genes encode protein products that can
be observed using certain biochemical
assays.
– β-glucuronidase (GUS)
– Luciferase gene
– Green Fluorescence Protein (GFP)