9.+10. Recombinant DNA engineering Flashcards
Explain what are restriction endonucleases
Restriction endonucleases: cut DNA at recognition sites in the middle of sequence (not the ends)
- bind to DNA non-specifically - slide until recognition site found - bind specifically
- often symmetric (palindromic) restriction sites (ex G/GAATCC)
- Mg2+ cofactor
- leaves cut termini: 3’ staggered / blunt / 5’ staggered
What are the enzymes used in recombinant DNA engineering?
- Type II restriction endonucleases
- Type II methylases
- DNA polymerase
- RNA polymerase
- Reverse transcriptase
- DNA ligase
- Exonuclease
- Terminal transferase
- Polynucleotide kinase
- Alkaline phosphotase
endo-exo
DNA-RNA pol
rev trans
lig-kin-transf
meth-ph
Explain what are restriction methylases
Restriction methylases: methylate restriction sites to prevent cutting by restriction endonucleases
-methylation added by:
- cognate methylase
- other DNA methylation patterns
BUT: DpnI cuts also methylated DNA restriction sites
Explain what are DNA polymerases
DNA polymerases: copy DNA from primer 3’ end
- synthesis 5’ -> 3’
- uses dNTPs
- DNA polymerases processive - catalyze multiple polymerisations
- 3’ to 5’ exonuclease activity - for proofreading (some also 5’ to 3’ exonucleases to remove DNA ahead)
Explain what are RNA polymerases
RNA polymerases: copy DNA sequence into RNA starting at promoter (transcription)
Explain what are reverse transcriptases
Reverse transcriptase: make DNA copy from RNA starting at 3’ primer
Explain what are DNA ligases
DNA ligases: joins two DNA backbone strands
- catalyse formation 5’-3’ phosphodiester bond (PO4 5’ + OH 3’)
- in DNA replication joins Okazaki fragments
- joins compatible sticky/ blunt ends
- T4 DNA ligase - rATP cofactor (forms intermediate with enzyme)
Explain what are exonucleases
Exonucleases: remove nucleotide residues from DNA ends
Explain what are terminal transferases
Terminal transferases: add homopolymer tails to DNA ends
Explain what are polynucleotide kinases
Polynucleotide kinases: add Pi from ATP to 5’ of DNA / RNA
- polynucleotide kinase: radioactively label 5’ of restriction fragments / ss oligonuclotides to use as probes
- T4 polynucleotide kinase: catalyses exchange of gamma position Pi from ATP to ss/ds DNA/RNA 5’ end (OH terminus)
Explain what are alkaline phosphotases
Alkaline phoshotases: remove terminal Pi from DNA ends
How are restriction endonucleases used as protection?
Some bacteria use restriction endonucleases as protection against bacteriophages - enzymes cleave DNA - destroy - no insertion
Bacteria protect own genome from those restriction endonucleases by methylating recognition sites - cognate methylases add methyl group - no cutting
What are the types of termini that restriction endonucleases could leave?
Cut termini could be:
- 3’ staggered
- Blunt
- 5’ staggered
What can prevent restriction endonucleases from cutting DNA?
Recognition site methylation prevents restriction endonuclease from cutting - methylation by methylases (cognate methylase)
How can ligation be manipulated to produce intra-molecular or inter-molecular ligation?
DNA ligase can join ends of different molecules / same:
- intra-molecular: add low DNA conc - joins itself -> circular DNA
- inter-molecular: add high DNA conc - joins diff molecules -> linear DNA
What type of ligation performed when two restriction fragments are linked into circular DNA molecule?
Both intra and inter-molecular ligation performed:
1) Inter-molecular joins one end of two linear molecules => linear
2) Intra-molecular joins two ends of one linear molecule joined => circular
Different types of DNA polymerases bbz ar reik nesamone
How can DNA polymerase be used to study restriction fragments or create detectable probes?
- DNA polymerase can add radioactively labelled alpha-dNTPs
- DNA polymerase can make radioactive DNA probe from hexanucleotide primers
What is a homopolymer tail?
Homopolymer tail: sequence added to blunt-ended polymers to join a ds DNA fragment into a cloning vehicle
What are the possible cloning vector sources for use in E. coli?
Vector sources:
- Plasmid derived - transfection
- Phage derived - transduction
- Combination of plasmid + phage - transduction
What are the common cloning vector properties?
Common vector properties:
- can promote autonomous replication
- can amplify from single copy
- have a genetic marker to select for / identify cells with vector (ex antibiotic resistance)
- restriction sites for inserting
- have minimal non-essential DNA
What are the examples of currently available cloning vectors?
Explain the process of molecular cloning
Process of molecular cloning:
1) Restriction enzymes for vector
2) Restriction enzymes for purified DNA - same type of termini (5’/3’/blunt)
3) Mix ligase + linear vector + target DNA at low conc (for inter-molecular ligation) -> will get vector+DNA / self-ligated vector
4) Transformation into E. coli
Explain the process of E. coli transformation with molecular vector
E. coli tranformation with vector:
1) Transform E. coli with vector by:
- CaCl2 + heat shock
- high voltage electroporation
2) Plate on + antibiotic plate
3) Only transformed E. coli will grow - have antibiotic resistance gene in plasmid
What can be used to prevent vector self-ligation in molecular cloning?
Phosphatases can be used to prevent self-ligation in vectors:
- exchange 5’ OH into Pi
- but vector and insert DNA can still ligate - only one Pi in the way - once one strand ligated - other can be ligated by E. coli repair system
Explain the process of recombinant clone screening by nucleic acid hybridization
Explain screening of recombinant plasmids using blue/white clony test
Blue/white:
inactive version of beta-galactosidase => white
alpha-peptide restores activity -> blue
How can vector and DNA insert be ligated if they cut with different restriction endonucleases?
Diff restriction endonuclease cut ends can be compatible:
if cut same by both endonucleases;
if ends are modified:
- use T4 DNA polymerase + dNTPs -> make blunt -> blunt end ligation
OR
- add DNA end inserts - ligate linkers with vector recognition site
Ex: EcoRI cut end can be ligated to SacI cut end
Explain how ends can be extended / shortened
DNA cut with restriction endonucleases can be modified to get needed sticky/blunt ends:
- extended: Klenow DNA pol I / T4 DNA pol for recessed 3’ ends
- shortened: T4 DNA polymerase has exonuclease activity - digestion of 3’ protruding ends
Explain how ends can be ligated using a linker?
Linker ligation strategy (blunt -> sticky ends):
- ligate multiple linkers - short blunt sequences added to ends of cut DNA fragment
- use restriction endonuclease to get needed ends
Explain how ends can be ligated using adapter ligation strategy
Adapter ligation strategy (blunt -> sticky ends):
- ligate adaptors - short DNA sticky sequences
- add polynucleotide kinase to add Pi to ends
What are K-12 E. coli strains?
K-12 E. coli - modified strain that doesn’t colonise human intestine - lacks O antigen
- EcoK restriction system degrades foreign DNA that is not methylated at specific sequence
- only hsdR- strain allows plasmid expression - restriction enzyme R is deleted -> plasmid not degraded (even though not methylated)
Smth more on K-12 E. coli
Explain the process of PCR
Explain what is a λ bacteriophage
λ bacteriophage:
- infects E. coli - used as a vector
- linear DNA - cos sites at both ends - cohesive ends - can circularise
- lytic + lysogenic pathways
What is λ bacteriophage DNA like?
λ bacteriophage DNA;
- linear - canc circularise
- cos sites
Explain lytic and lysogenic λ bacteriphage cycles
Lytic:
- infects bacterium - rolling circle replication - mutiple copies - capsid proteins produced -> new bacteriophage assembly -> burst
Lysogenic:
- infects bacterium - integrates into chromosome -> replicates
Explain rolling circle replication
Explain rolling circle replication in λ bacteriphages
Explain the process of λ DNA packing into bacteriophages in vitro
λ bacteriophage packaging in vitro:
1) E. coli lysed - contents out
2) Ligated λ DNA catenanes are cut in vitro by Ter at cos sites - genes for packaging
3) From lysates proteins assemble + package λ DNA -> bacteriophages assembled
How can λ be converted into cloning vector?
λ can be converted into cloning vector:
1) Can’t simply insert into λ genome - will be too large to fit inside phage head -> cut out non-essential region
2) Inserts possible - insert stuffer region (restriction sites) - vector made
3) λ phage assembly in vitro => infections recombinant λ phages
What is the λ genome map?
λ genome:
- essential region: capsid components, early/late regulation, DNA synthesis, host lysis
- non-essential region: integration and excision
What is a replacement vector?
Replacement vector: λ DNA cloning vector produced by cutting out non-essential regions and inserting cloning genes - possible because of λ phage packaging mechanism
λ vetor making pic phage assembly in vitro
What is a cosmid vector?
Cosmid vector - plasmid with λ cos site ligated with new ~35Kbp DNA inserts
High conc ligation produced catenane structure for in vitro packaging
Efficient in bacteria infection - in cells cosmid vector + DNA insert circularise via cos - replicated as plasmid (Amp+)
What are the differences between λ vector and cosmid vector?
λ vector:
- bacteriophage
Cosmid vector:
- designed plasmids - bacterial oriV - antibiotic selection marker - cloning site - cos sites from λ bacteriophage
What is λ phage catenane structure?
??
How can a gene from a complex organism be cloned?
Cloning a gene frmo complex organism:
1) Construct genomic / cDNA library (contains representation of all DNA/RNA in starting material - screening the library to identify desried clone)
2) PCR to aplify specific sequence in genomic DNA/ cDNA (reverse transcription)
3) Transfect bacteria
What is the difference between genomic DNA libraries and cDNA libraries?
Genomic library: all genes of an organism
cDNA library: genes expressed by a **specific cell **type in an organism (constructed from expressed mRNAs - reverse transcriptase-> DNA)
How is a genomic library constructed?
Genomic library is constructed cloning overlapping DNA fragments generated by partial digestion of genomic DNA with restriction enzymes (cut to be compatible with vector)
Why DNA fragments are overlapping in genomic DNA libraries?
In genomic DNA libraries DNA fragments overlap because xxx??
How is a genomic library made in a λ replacement vector?
Explain λ library plating
How is a genomic library made in a cosmid vector?
How to calculate the number of individual clones required in a genomic library to approach complete coverage?
Explain what is chromosome walking
Chromosome walking: hybridization of one library clone to another can find overlapping regions of DNA, and hence can be used for identifying upstream and downstream sequences from the marker
One probe binds - polymerization - site for another probe
What is a BAC vector?
Bacterial artificial chromosome (BAC): an engineered derivative of E. coli F factor
- replicate 1, 2 copies per cell
- stable maintenance for their DNA inserts
- best for genomic lib construction with inserts of 200-300 Kbp
- once insert is ligated into BAC - electorporated into E. coli
Explain BAC colony plating
Explain the process of cDNA production
1) mRNA purified
2) Oligo-dT tail added (complimentary to polyA tail)
3) Reverse transcriptase used to make complementary DNA to RNA
4) RNA degraded RNase
5) DNA pol used to synthesise second complementary DNA strand
How can cloning efficiency be improved?
1) Methylate DNA
2) Add restriction enzyme sites using linkers
3) Cut with restriction enzymes => better ends for ligation into vector
How can cDNA fragments be cloned into λ insertion vectors?
?? on cDNA mentioned
How can a cDNA library be screened for specific clones?
Ways in which cDNA libraries can be screened for clones:
- hybridization - DNA/RNA oligo nucleotide probes
- PCR
- Using Ab for expressed protein
- functional cDNA screens in specific cell types
Explain cloning PCR amplified DNA
How can gene clones be used in research?
To identify genes using related species amplified genes as probes
What is a multi-component plasmid?
Multi-component plasmid: plasmid with inserts form different sources
How can expression of recombinant DNA be visualised?
Reporter expression
- reporter incorporated into vector along with inserted fragment
- reporter genes - ex GFP
- reporter genes can be expressed:
1) directly from promoter
2) as part of a fusion protein
3) linked to promoter-cDNA expression unit (via IRES)
What is a shuttle vector?
Shuttle vector: vector that can propagate in two different host species - inserted DNA can be tested in two different cell types
Explain functional cDNA screening for a particular gene
cDNA screening: screening libraries of cDNA clones rely on the selective binding of nucleic acid probes to cDNA / Ab to gene protein product encoded by the cDNA
What is site-directed mutagenesis of cloned DNA?
Site-directed mutagenesis: specific nucleotide changes (deletions or insertions) of single / several nucleotides in cloned DNA
What could be the outcomes of site-directed mutagenesis of cloned DNA?
Site-directed mutagenesis of cloned DNA could result in:
- changed am a sequence
- a restriction site
- altered gene regulatory regions
What are the two DNA construct assembly strategies?
DNA construct assembly strategies:
- Golden Gate assembly: Type IIS restriction endonucleases - ligation in vitro
- Gibson assembly: DNA joined via annealing in vitro
Explain ‘Golden Gate’ assembly strategy
‘Golden Gate’ assembly:
1) Type IIS restriction enzyme cleaves away from recognition site - leaves staggered ends
2) Position flanking sequences - DNA can be cut to create compatible staggered ends
3) Ligation
How can restriction sites for ‘Golden Gate’ assembly be introduced into vector?
BsaI sites introduced by incorporating them into oligo PCR primers
Overview of ‘Gibson’ assembly strategy
‘Gibson’ assembly: independent seamless joining - ends edited - joined
Explain ‘Gibson’ assembly strategy
What was ‘Gibson’ assembly originally created for?
‘Gibson’ assembly was create to generate synthetic genomes
Lecture overview
