Sequence-based cloning approaches Flashcards
What are 4 things in the traditional molecular cloning toolkit?
Restriction enzymes
DNA ligase
DNA polymerases
Alkaline phosphatases
What are restriction enzymes/restriction endonucleases?
- cleave duplex DNA into fragments
- blunt/sticky ends
- type 2 - cleave within recognition site (4-8bp)
What is DNA ligase?
- form recombinant DNA - joins compatible ends of duplex DNA
- from T4 bacteriophage
What are DNA polymerases?
- Klenow fragment - lacks 5’-3’ exonuclease
- used to fill in 5’ overhangs in presence of nucleotides
- klenow trims 3’ overhands to form blunt ends
What is alkaline phosphatase?
- catalyses the removal of 5’ phosphate groups
- prevents DNA ligase from making phosphodiester bond
What are plasmid-based vectors?
Natural plasmids
replicate independently of chromosome
carry antibiotic resistance gene
What is another term for a poly linker?
Multiple cloning site
what are the 3 stages of cloning DNA with plasmid vectors?
1) Generation of recombinant molecules
2) Introduction of recombinant molecules into host bacteria
3) Selection of “transform ants”
what are the details of stage 1 of cloning DNA with plasmid vectors (Generation of recombinant molecules)?
linearise with restriction enzymes
dephosphorylate with a phosphatase to prevent vector religation
ligate vector with fragment to be cloned - must have compatible ends
What are the details of stage 2 of cloning DNA with plasmid vectors (introduction of recombinant molecules into host bacteria)?
introduce DNA into host bacteria by chemical transformation (CaCl2) or electroporation
most bacteria do NOT take up plasmid - need to select!
What are the details of stage 3 of cloning DNA with plasmid vectors (selection of transformants)
Plasmid replication and bacterial cell division
spread o ampicillin plates
- cells taken up plasmid (AMPr) form colonies
What is the use of Xgal?
chromogenic substrate
hydrolyses if beta galactosidase is produced -produced insoluble blue pigment
added with IPTG for blue- white screening
What is IPTG?
analog of galactosidase - non metabolising but can induce expression of LacZdeltam15 gene in chromosome
What are 3 key features of the bacterial host?
disablement - us auxotroph
stable maintenance of transformed DNA
efficient transformation by plasmid DNA
What is genome annotation?
process of identifying the locations of genes within sequences of chromosomes - provide details of their molecular function and cellular role
What is the use of primers with 5’ extensions?
sequences at 5’ end that will not be involved in base-pairing in 1st round PCR
incorporated from 2nd round on
Facts about Golden gate assembly?
single tube reaction - BsaI, DNA ligase
assembly of a vector and 1 or more DNA fragments into constructs suitable for direct transformation of a bacterial host
scarless
no recognition sites retained
What is BsaI?
type IIS enzyme
non-palindromic
cuts outside recognition site
4 nucleotide overhang
can create all 256 combination
What is the life cycle of bacteriophage lambda?
adopts alternative lysogenic state - genome of lambda become incorporated into genome
What is DNA present as in phage particles?
linear dsDNA
single stranded terminal i of 12nt - complementary to each other
How can nicks in E.coli be closed?
DNA ligase
What is lysis in the lambda life cycle?
DNA replication
synthesis of phage products
particle assembly
cell lysis
What is lysogen in the lambda life cycle?
integration of lambda into host genome
cells immune to further infection
How is liquid culture assay carried out?
phage stock added to bacterial culture
bacterial lysis and then sometimes lysogenation occurs
How is plaque assay carried out?
phage stock added to bacterial culture
aliquot spread on surface of agar plate
lysis and lysogen - turbid plaques
How are infective phage particles formed?
DNA between cos sites are packaged into heads and associate with preformed tails to produce infective particles
What is the DNA length that is packaged into a phage head?
78-105% of wild type lambda- 48.5kb
parts of lambda can be removed - increasing size of DNA that can be cloned
What are lambda replacement vectors?
central non-essential region substituted with stuffer fragment
stuffer allows propagation in E.coli
cloning - stuffer removed and replaced by foreign insert
Breaking the 22kb barrier or lambda vectors?
possible to clone larger fragments - COSMIDS
How to make a cosmid library?
propagate cosmic vector as PLASMID
cleave with restriction enzyme, add foreign DNA, ligate to form concatemers, not circular products (linearise)
How do recombinant cosmids circularise?
via cos sites on entry into host bacteria
What genes to recombinant cosmids lack?
genes required foe lambda parcel production
What are limitations of traditional cloning?
separate reactions
restricted to joining of only 2 DNA fragments, vector + insert, in single ligation
additional rounds needed to join additional fragment
can be limited by availability of suitable restriction enzyme sites
joining of large fragments problematic due to lower concentration of ends
What are the benefits of Gibson (Isothermal assembly)?
assemble multiple fragments in single reaction
long fragments
scarless
any insert with appropriate overhangs can be used
What is the process of Gibson assembly?
5’-exonuclease activity produces 3’ overhangs
3’ overhangs complementary base pair - produce pairs of primer and template
self-priming reaction, DNA polymerase activity extends from 3’ end of overhangs now annealed to complementary DNA to fill in the gaps
DNA ligase seals the nick - covalently links DNA fragments together
How is it possible to join fragments of only one orientation and order by Gibson assembly?
use different sequences for each pair of ends to be joined
What are some other cloning approaches?
Gateway recombination
TOPO
Ligation-independent
Yeast-mediated cloning & oligonucleotide stitching
Cloning by order
