Cloning of DNA Flashcards
What is DNA cloning
technique involving insertion of a foreign DNA fragment into a vector capable of replicating separately in a host cell
what allows multiple copies of identical DNA insertion
Growing the host cell allows the production of multiple copies of identical inserted DNA for use in a variety of purposes
what is the host cell
a cell that has been introduced with foreign DNA or RNA and carries it around
examples of methods used for DNA cloning
- cloning by restriction enzyme digestion and ligation of compatible ends
- T- A cloning directly from a PCR product
- TOPO- attached unidirectional cloning.
- recombination based cloning
what is T-A cloning directly from a PCR product
does not require restriction enzymes
add Adenine by PCR to 3’ ends
it relies on the ability of Adenine and Thymine on different DNA fragments to hybridize; the ligase binds them together
what happens to the vector in T-A cloning
vector linearized to make blunt ends, ddTTP added to 3’ ends
what is TOPO cloning
Vaccinia virus topoisomerase I recognises the DNA sequence 5’ –(C/T) CCTT-3’ in supercoiled DNA during replication
enzyme digests, unwinds and re-ligates the DNA at the 3’ phosphate group of the T base
what is TOPO - attached unidirectional cloning
- design PCR primer with CACC at its 5’ end (no modification of 3’ Primer is necessary)
- amplify with proofreading polymerase
- mix PCR product with vector, incubate 5 min, and transform E. coli
form intermediate construct and extra sequence cleaved off in E.coli, form directionally cloned PCR product
what is recombination based cloning
takes advantage of the site-specific recombination reactions enabling the bacteriophage λ to integrate and excise itself in and out of a bacterial chromosome
what are the requirements for DNA cloning
- foreign DNA
- host organism
- vector DNA for cloning
- means of inserting foreign DNA into the vector
- method of placing the in vitro modified DNA into the host cell
- methods for selecting and/or screening cells that carry the inserted foreign DNA
what must the foreign DNA be like for DNA cloning
PCR product
genomic DNA
complementary DNA (cDNA)
what does the host organism require
Bacterial host – E. coli
Eukaryotic host – yeast, mammalian or plant cells
other hosts – insect cells, etc
what is complementary DNA
DNA synthesized from an mRNA by reverse transcriptase and DNA polymerase
what is dsDNA
double strand DNA
what is ssDNA
single strand DNA
what is a vector DNA
DNA molecule that functions as a “molecular carrier” that brings the DNA of interest into the host cell & facilitates its replication
what are the requirements for a ligation reaction
- two or more fragments of DNA (blunt/cohesive)
- buffer containing ATP
- T4 DNA ligase
how is the DNA inserted into the vector to be cloned
foreign DNA must be ligated into the linearized vector
what is transference of DNA into the host cell
method of placing the in vitro modified DNA into the host cell
what occurs in a transformation event
- bacterial cells take up naked DNA molecules
- cells need to be made “competent”
- cells are heat-shocked
- shock allows the molecules to get inside cells
- good efficiency of transformation ranges between 107 to 108 colonies/μg DNA
what is another method for DNA transfer
Electroporating the DNA into the host cell
what occurs in electroporating
- membranes of cells are permeablized using an electric field
- DNA present can penetrate the membranes after the electric shock
- protocols differ for various species
- efficiencies of transformation ranges from 109 per μg DNA (3 kb) to 106 (136 kb)
what is conjugation
- natural transmission from donor to recipient
- occurs with a host cell that is not readily transformed
- formation of cell to cell junctions
what is transfection of DNA
- DNA is packaged in vitro into phage particles
- phages are allowed to infect bacterial cells
- DNA is transiently expressed
what occurs in selecting and screening recombinants
Once the DNA of interest has been cloned, transformed cells carrying it must be identified
what are the selective conditions that hosts are grown in
- antibiotics
- nutrient requirements
- plaque type
- blue-white selection (β-galactosidase)
- specific (hybridization, antibodies, PCR)
why are hosts grown in selective conditions
allow the identification of positive clones containing the carrier vector with the DNA of interest
what is a selectable marker
a gene introduced into a cell that confers a trait for artificial selection
what is phenotypic screening
protein encoded by the gene changes the colour of the colony
what occurs during blue-white screening
- lacZ gene – encodes for β-galactosidase
- X-Gal – substrate for the enzyme [X-gal is white but if -galactosidase is present, substrate blue]
- IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer
- cloning sites within the lacZ gene
- disruption of gene by insertion of the foreign DNA
what occurs in the DNA ligase mechanism
Energy needed for adenylation of DNA ligase reaction ATP loses a Pi AMP Release a molecule of water Joining of DNA segments
what are the blue colonies like in the blue-white screening
blue colonies have a functional β-galactosidase protein
what are the white colonies like in blue-white screening
white coloines have a non-functional β-galactosidase protein
in antibiotic resistance what plasmid does bacteria need to be resistant to Kanamycin
Kan ^r
how do you read a plasmid
like a clock
what is the bla gene for in a vector
resistance to plasmid
what is the lacZ for in vectors
gives blue colour
what is the rep gene for in vector
helps plasmid replicate
what is the MCS polylinker
multiple cloning site
what happens in blue-white screening when an insertion occurs
will get a white colour as no functional beta galactosidase protein due to insertion
how does large scale propagation work
once colonies are identified, they are cultured in broth to increase numbers and therefore the amount of DNA
how are colonies stored
samples are also prepared for storage at -80 degrees C
they can be kept for many years
how is a segment of DNA cloned
bacterial plasmid with ampicillin resistance, lacZ gene and restriction site
isolate plasmid (vector) DNA and human DNA
insert human DNA into plasmids
add DNA ligase to bond covalently
put plasmids into lacZ bacteria by transformation
clone cells
identify clone carrying gene of interest
what must happen to the DNA used in DNA cloning
cut both DNAs with same restriction enzyme so have sticky ends
mix DNAs, join by base pairing
how are cells cloned in DNA cloning
plate cells onto medium with ampicillin and X-gal, identify clones of cells containing recombinant plasmids by their ability to grow in presence of ampicillin and their white colour
why would we want to be able to clone
isolate DNA
establish collections
for further molecular studies
why do we need to be able to isolate DNA
can then be used for making probes, to be digested, to be mapped and/or to be sequenced
what could further molecular studies be
Production of special proteins
Investigation of protein/enzyme/RNA functions
Identification of mutations e.g. gene defects related to specific diseases
what are the methods to clone DNA
- restriction enzyme digestion and ligation
- T-A cloning
- TOPO attached unidirectional cloning
- recombination based cloning
what does DNA cloning require
segment of DNA, a host, a vector, a means to insert DNA into the vector and the vector into the host, and a selection method for the segment of interest
why is DNA cloning useful
fundamental for recombinant DNA technology
