The technology underpinning molecular biology: gene cloning Flashcards
For what downstream purpose is cloning genes necessary?
to study, modify or transfer a particular gene (or fragment of DNA
What is gene cloning?
identification and replication of DNA at a high copy number, in a host or in vitro
To be replicated in a host, DNA must
be a replicon
replicon
contain a replication origin
Most DNA fragments are not replicons & thus
would be diluted during host cell division
How is the replicon issue solved?
- plasmids and bacteriophages are replicons
- used as “vectors”
- enable replication of a single DNA fragment in a host cell (or clone of cells)
- generate a library of many cells/clones, each one containing a different fragment
Describe the function of a gene library
screened to identify the clone carrying the DNA of interest
Describe PCR
used to replicate DNA at a high copy number in vitro
Describe gene cloning
- construct different recombinant DNA molecules using DNA fragments
- introduce into a bacterial host
- plate out onto an agar medium, to grow colonies or clones of identical host cells
- grow at 37 degrees
- each colony contains multiple copies of just one recombinant DNA molecule
Give an example of a bacterial host used in gene cloning
E. coli
Describe phage resistant bacteria
- host DNA is modified and therefore protected, by cleavage phage DNA
- no plaques form
- restricting bacteria - endonuclease
Describe susceptible bacteria
- phage offspring released then cell is injected with phage DNA by bacteriophage
- cycles of cell lysis and localised reinfection form plaques
How is DNA cut with restriction enzymes?
- palindromic recognition site (4,6 or 8bps long)
- binding
- cleavage generates cohesive or blunt termini
Describe the relationship between recognise site length and cut frequency
- 4 bp cutters cut more frequently
- 8 bp cutters cut less frequently
Give the name of a restriction endonuclease
EcoRI
cohesive termini
sticky ends
How is the cut DNA joined?
- hydrogen bonds form between cohesive termini
- phosphodiester bond formation catalysed by DNA ligase in ATP-dependent reaction
Describe the DNA ligase molecule used in cloning
- T4 DNA ligase
- typically from bacteriophage T4
What is the function of gel electrophoresis?
separating DNA fragments based on size
How does gel electrophoresis work?
- agarose gel immersed in buffer, placed in an electric field
- DNA samples loaded into wells at one end of the gel
- fragments migrate towards the anode
Describe intercalating agent in gel electrophoresis
- e.g. EtBr
- inserts itself between DNA bases and fluoresces
- can be viewed under UV transilluminator
Describe the allele ladder
- DNA fragments of known sizes, purchased commercially
- used to size the fragments of interest
What are the two types of libraries?
- genomic library
- cDNA library
Describe a genomic library
- used if interested in the whole transcription unit or intergenic regions
- contains all the DNA sequences in a cell
- can be >90% non-coding
- sample DNA must be fragmented before cloning
- fragment / insert sizes typically large (e.g.~15-20 kb if using phage vector)
- high number of repetitive sequences
- size of library required depends on genome size
Describe DNA fragmentation before cloning in a genomic library
- neither practical nor helpful to clone an entire chromosome
- partial restriction enzyme digestion or random shearing
What does the quality of a genomic library depend on?
- quality of fragmentation
- need maximum number of large, overlapping fragments
Describe a cDNA library
- required if only interested in mRNA sequence
- made from mRNA of a particular tissue-type or time-point
- inserts are DNA, but are complementary to the mRNA
- sample fragmentation not required
- fragment / insert sizes typically small (~0.5-5 kb)
- may not contain all gene sequences
- contains few repetitive sequences
- size of library needed depends on relative abundance of mRNA of interest