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
What affects the quality of a cDNA library?
- quality of the mRNA isolated
- gene expression profile of sampled tissue
When would a plasmid vector be chosen?
- 5,000bp insert size
- 1,800,000 clones to represent human genome
When would a phage vector be chosen?
- 20,000bp insert size
- 450,000 clones to represent human genome
When would a cosmid vector be chosen?
- 40,000bp insert size
- 225,000 clones to represent human genome
When would a BAC vector be chosen?
- 150,000bp insert size
- 60,000 clones to represent human genome
When would a YAC vector be chosen?
- 300,000bp insert size
- 30,000 clones to represent human genome
cosmid
- plasmid with cos sites
- allows packaging into phage particles
- more efficient delivery
BAC
-bacterial artificial chromosome
- based on F plasmid that can carry v. large inserts at low copy no.
YAC
yeast artificial chromosome
Describe plasmid vectors
carry inserts up to 5-10kb
Describe puC18/19
- encodes beta-galactosidase
- ori, MCS, lacZ, AmpR
- creates blue product from colourless X-gal
- allows for blue-white selection on an X-gal plate
AmpR
selectable marker
Describe phage vectors
- carry inserts up to 20kb
Describe phage lambda
- 48,502nta
- 20kb dispensable, stuffer fragment required for lysogen, but not important for vector use
- 12bp cohesive ends at each arm enable packaging and circularisation in host
- creates plaques
Describe YAC vectors
- shuttle vectors that can be propagated in bacteria to prepare vector for cloning, or in yeast to carry large inserts
- carry inserts up to 300kb
Describe circular YAC vector
- e.g. pYAC3
- cloning site (EcoRI) - inserted here
Describe the preparation of a genomic library in a phage
- genomic DNA partially digested with restriction enzyme
- stuffer fragment removed from bacteriophage lambda
- genomic fragments ligated into vector arms
- concaterner of many recombinant phages cleaved
- packaged into infective phage particles
- infected into E. coli
- plaques develop
Describe the results of a phage genomic library
- each plaque correspond to a single clone
- each clone carries a single fragment of genomic DNA from the genome of interest
Describe the purification of mRNA for cDNA synthesis
- total cellular RNA isolated from cells and passed through elution column
- poly-A tails of mRNA pair with oligo(dT) chains
- mRNA retained in column
- other RNAs pass through
Describe total cellular RNA
- prepared by treatment of cell lysate with organic solvents to remove protein/DNA
- or using a commercial kit
Elution column
contains oligo(dT) chains linked to cellulose
oligo(dT)
short, single-stranded DNA sequence containing only thymine nucleotides
cellulose
inert matrix
mRNAs
have poly-A tails
When you purify RNA from cells, it is
a complex mixture containing rRNA, tRNA and mRNA et al.
What is the function of the buffer in the elution column?
breaks the hydrogen bonds between poly-A tails and oligo(dT) chains
cDNA synthesis requires
- purified mRNA
- dscDNA
Describe the first strand of cDNA synthesis
- mRNA template
- oligo-dT primers added and anneals
- 4x dNTPs and reverse transcriptase added
- incubation
- creates sscDNA
dNTPs
nucleoside triphosphates
Describe the second strand cDNA synthesis
- RNaseH generates nicks in mRNA
- second strand synthesis begins with DNA polymerase I
- second strand conclusion with T4 DNA ligase
DNA polymerase I
exonuclease
Why are library plate replicas required?
- easy to screen
- viable master plate can be preserved for future propagation
Describe the replication of library plates
- nitrocellulose disc overlayed onto master plate containing clone library inside E. coli cells
- binds DNA efficiently to make a replica
- disc removed and cells lysed with NaOH (denatures DNA)
- baked and treated with UV light
- DNA strands bind to disc
- ready for screening
How are probes made?
- labelling of a DNA fragment
- purified DNA corresponding to, or similar to fragment of interest forms a template
- denatured by heating to 95 degrees, then following
- mixture of random hexanuclotide primers anneal
- add labelled dNTPs and Klenow fragment of DNA polymerase I
- denatured using heat to form a ss
- incorporation of labelled nucleotide means that it is a probe
Name some ways of labelling dNTPs
- radioactive
- fluorescent
- enzyme-linked detection tag
Describe radioactive screening by DNA hybridisation
- DNA on replica nitrocellulose filter
- radioactive probe added
- replica hybridised, washed and dried
- labelled probe forms duplex with cDNA
- autoradiography
Describe autoradiography
- exposure to X-ray film
- detects radioactivity
- spot on film locates colony
- related to the master plate to identify the clone of interest
- clone propagated
Describe PCR
- DNA is heated to 90-100 degrees to separate the two strands
- DNA quickly cooled to 30-65
- short, ss primers anneal to complementary sequences
- heated to 60-70 degrees
- DNA polymerase synthesises new DNA strands
- two new, dsDNA molecules created
- cycle repeats, doubling DNA each time
What are the ingredients necessary for PCR?
- template DNA
- 2x ssDNA oligonucleotide primers
- 4x dNTPs
- taq polymerase
- thermal cycler
From where is taq polymerase derived?
Thermus aquaticus
Describe PCR
- much faster than library
screening - requires prior knowledge
of target for primer design - in vitro method
- products can be cloned in
vectors for further propagation
and manipulation
How long does library screening take?
several weeks
