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
