DNA Cloning Flashcards
What is cloning?
A sequence of DNA into a plasmid vector. this vector can then be put into a cell
What is the aim of genes cloning?
the aim of gene cloning is to obtain isolated and purified copies of specific gene sequences
cloned genes allow us to:
- determine the sequence of the gene (and protein)
- obtain leads into the function of the gene
- manipulate the gene
What are the two pathways for gene cloning to take?
1) to understand the gene / protein
2) exploit the gene
clone
a large number of identical cells or molecules with a single ancestral cell or molecule
DNA cloning vector
a carrier DNA molecule that allows attached DNA to be replicated in a cell
e.g. plasmids or viruses
Restriction enzymes
an enzyme that recognises and cleaves DNA at specific sequences
e.g. EcoRI cleaves the palindrome GAATTC
DNA ligase
an enzyme that can covalently link DNA molecules
What are Plasmids?
Double stranded DNA molecules that replicate in cells independently of the host chromosome. They are inherited by daughter cells and are no-essential for growth
Why are plasmids an ideal cloning vector to use?
- it is well characterised (sequence ad function of genes known)
- small and easy to purify and manipulate
A plasmid vector must contain:
- an origin of replication (Ori)
- a selectable marker
- unique restriction enzyme cleavage sites
The Four steps that is involved in cloning a piece of DNA
1) Restriction
2) Ligation
3) Transformation
4) Selection
Restriction
the plasmid vector and the DNA fragment of interest must be digested with a restriction enzyme to produce sticky end
Ligation
the DNA fragment of interest must be ligated into a plasmid vector
Transformation
the ligated plasmid vector must be transformed into a bacterial host (usually E.coli)
Selection
bacterial colonies carrying the plasmid with the DNA fragment must be selected from those that do not
What enzyme does the lacZ alpha gene encodes?
beta-galactosidase alpha peptide
If Beta-galactosidase is functional?
a bacterial colony that contains an active beta-galactosidase will cleave x-gal which means that it will produce blue colonies
If Beta-galactosidase is non-functional?
a bacaterial colony that does not have an active beta-galactosidase means that it cannot cleave the x-gal which means that it will not produce any colour change so the colonies are white
IPTG relation with lac Z gene?
if IPTG is added means that the lacZ is always being made
what advantage does adding a promoter in front of the MCS?
this would allow for expression of the gene we have cloned in
control of expression
Ptac promoter - induced by IPTG
ccdB
toxic cells
Amplification
a process by which nucleic acid molecule is enzymatically copied to generate a progeny population with the same sequence as the parental one
PCR
to amplify a specific sequence of a DNA
Genomic library
contains DNA fragments representing the entire genome of an organism
cDNA library
contains cloned cDNA (reversed-transcribes mRNA), representing the transcriptome of an organism
DNA inserts are stored in plasmids or BACs:
Plasmids
- insert size <20kb
Bacterial artificial chromosomes
- insert size 75-300kb (fewer clones required)
