Cramming Flashcards
What are the examples of histochemical tests?
LacZ and UidA
What is an example of bioluminescence?
Lux
What are the essential features of all vectors?
Ability to replicate in host
Ability to undergo efficient transformation
Ability to take up recombinant DNA (insert foreign vector)
How does the host ensure efficient transformation by the plasmid?
Use a host deficient in natural restriction modification
E.Coli K strain - hsdRMS encodes K restriction-modification system so delete hsdR gene
How does a host ensure it is disabled?
Use an auxotroph - can only be used on a growth medium in the lab
How do you maintain stable maintenance of the transformed DNA?
Avoid re-arrangements by using mutants in recombination genes e.g. RecF and RecA
What is the insert range for plasmids?
1-10kbp
What type of DNA is in a lambda vector?
Linear double stranded
What are the complementary termini in a lambda vector for?
Can join to each other and circularise
What are the two stages of lambda life cycle?
Lytic and Lysogeny
What happens in liquid culture when the virus undergoes lytic replication?
Turns pale
In plaques - area of dead cells that appears lighter than the plaque turbidity
What is a prophage?
Temperature phage genome incorporated into host chromosomes
What is the packaging constraint in lambda?
78-105% of DNA (48kb)
When making an insertion vector what can you not delete?
More than 25% of the wild type lambda
What is the insert limit for insertion vectors?
0-10kb - no lower limit
What do insertion vectors generate when testing for their presence?
A plaque
How are replacement vectors made?
A stuffer fragment full of junk DNA is used to fill in the non-essential segment that is removed
What size fragments can an insertion vector carry?
Up to 22kb
What will in vitro packaging extracts pack?
Any molecule that carries cos sites separated by 37-50kb
What are cosmids?
Vectors that contain the lambda cos site
How are foreign inserts inserted into cosmids?
Cleave with restriction enzymes and add foreign DNA and ligate to form concatemers
What does cosmids lack?
Genes required for lambda particle production
When and how do lambda particles circularise?
On entry into host bacteria via the cos sites
What type of phage is MI3?
Filamentous
What is the genome of M13?
Single stranded (6.4kb)
What are large inserts in M13 prone to?
Rearrangments
What specificity are M13?
Male specific - they need the host to express the F plus before being internalised
What do M13 produce in culture?
Plaque - infected cells release more phage which then infects neighbouring cells
Where do you isolate double stranded DNA from and single stranded DNA for the M13?
Double - pellet
Single - released from the phage into the supernatant
What is the insert size range for M13?
0-10kb
How is phage display created?
New genetic material is inserted into a phage gene, bacteria process the new gene so that a new protein or peptide is made - each phage receives a different gene collectively the population of phage can display a billion or more proteins or peptides each ties to its own gene. The proteins keep most of the physical and chemical properties of their parent proteins - the library is exposed to an immobilised target, some will bind the target and others will not.
How do you make a genomic library?
A collection of clones which together contains copies of all the nuclear DNA of the organism
What are the steps in making a genomic library?
1) Preparation of insert genomic DNA
2) Fragment genomic DNA to suitable size for ligation into vector (20-25k for replacement and 45kb for cosmic - Sau3A for partial digestion)
3) Ligate purified insert DNA
4) Package recombinant concatemer into phage particles in vitro
How big is the mammalian genome?
3x10^9
How do you detect clones by molecular hybridisation?
Cloned recombinant DNA molecules are denatured and the single strands are attached to a nitrocellulose membrane.
The membrane is then incubated in a solution containing a radioactivity labelled probe that is complementary to some of the nucleic acid bound to the membrane.
Alkaline solution - disrupts the virions, releasing and denaturing the encapsulated DNA.
What are the uses of gene libraries?
Substrates for genome sequencing projects
Sustrate for PCR
Genetic tests
Functional genomics
Outline the modern method of DNA sanger sequencing.
dsDNA template DNA denatured with a thermal cycler
Primer oligonucleotide
Thermostable DNA polymerase
dNTPs
Fluorescently labelled ddNTPs
One pot reaction - multiple rounds per template enhances yield
Capillary electrophoresis for separation
Laser excitation and filtered fluorescence for detection
Detects each fluorescence as it passes through a detector
Outline Illumina sequencing.
dNTP analogus are blocked at the 3’OH end and fluorescently labelled - a mixture is added to the growing chain but one base will be added and then detection takes place - the labile bonds are cleaved removing the fluorophore and the process continues.
Happens on massively parallel scale - amplification of signal
What type of polymerase is taq?
Klenow fragment
What do Kod and pfu possess?
3’-5’ exonuclease - proof reading
What are you mutating in galactose oxidase?
TGG to GGC
What vector was originally used for site directed approaches?
M13 - single stranded
What were the problems with early site directed experiments?
Poor ligation
Strand displacement
Mismatch repair
Mutant recovering
What is the Tm of the primers in quikchange?
78
What does Dpn1 digest?
Methylated DNA - parental. Without this lots of your products would be parental DNA.
What type of amplification is quikchange and why?
Linear - the daughter strands can’t be amplified due to the nick so only the parents carry on being amplified
What E.coli cells are the daughter strands put into?
XL10 Gold E.coli cells
What is cassette mutagenesis?
Insert a section of DNA between two restriction sites - need the vector to have restriction sites in a suitable place. The primers overlap forming complementary ends to the restriction site. The primers are also phosphorylated to improve efficiency.
What are the two primers used in PCR mutagenesis?
One is complementary and the other contains the mutation
Typically 100bp long fragment to insert
What type of template does sticky feet mutagenesis use?
Normally single stranded but it can work with double stranded.
Large insertions >100bp.
What are the different types of fluorescent filters?
Bandpass: 630nm
Longpass: 520nm
Shortpass: 575nm
What are the different light sources?
Hg vapour burners, xenon burners, metal halide, LEDs and lasers
What are fluorescent derivatives?
Xanthene, cyanine and pyrene
What are the biological applications of fluorescence?
Fluorescent microscopy Flow cytometry Real-time PCR Conjugation to antibodies and modified nucleic acids Immunofluorescence assay
What are the different colours of GFP?
Yellow, blue, orange (red is synthetic)
How can you optimise codon usage in E.coli?
Some E.coli strains have additional tRNA genes to enhance expression of these genes - can choose this strain.
Mutate critical codons to more commonly used codons
Resynthesise the complete gene to reflect host codon usage
Why use fusion tags?
Allow easy identification for improves stability and folding
Either C or N terminal
Hisitidine tag or streptag
How does a hisitidine tag work?
Load onto a column with nickel ions (NTA resin) - wash to remove non-specific binding and elute with imidazole - same side chain so competes with higher affinity
What is a streptag?
Bind to streptactin via streptag (WSHPQFEK) - mimics biotin. Competed of by desthiobiotin
What happens in maltose protein fusions?
Amylose affinity column
Protease site between the MBP fusion and the target recombinant - after purification it can be cleaved with protease and then the mixture can be passed through an affinity column again to remove the MBP component allowing the recombinant to flow straight through and be collected
What are tac and lac repressors induced with?
IPTG
What are pBAD?
Arabinosr or rhanmose promoter - promotes response sensitivity to concentration of arabinose or rhamnose in growth medium and so increasing concentrations lead to increased expression.
How can you overcome the effects of steric hinderance by secondary structures?
Change the sequence of the coding region - silent mutations
What are the advantages of yeast expression system?
Flexibility - high or low copy number plasmids, inducible or constitutive promoters are available
Some eukarotyic post-translational modifications
Deletions of genes for homologous proteins allow functional assays in vivo by complementation
Multiple plasmids can be maintained
Cheap and easy to grow
Well described cell biology and genetics
What are the two examples of yeast expression systems?
Pichia pastoris: AOX1 promoter, independent plasmid replication - glycosylation may be a problem
Sacchromyces cerevisiae: autonomous or integration
What is the tm?
The temperature at which have strands are denatured and have are double stranded
What does low salt do to hybrids?
Destabilises them
What does nick translation use?
alpha-32P and DNA polymerase
Label the alpha part of ATP
Add DNase I to get some digestion and then fill in with nucleotides
What is primer extension?
You have lots and lots of randomly labelled hexameters (4^6 different sequences) - at least a few of these will anneal to the probe DNA sequence
Denature the DNA template and anneal primers
Use a DNA polymerase without 3’-5’ exonuclease else you will digest the primer
What are riboprobes?
Probe fragment is cloned into a vector containing specific phage promoters - recognised by the corresponding RNA polymerase and translated
Use NTP’s as RNA - incorporate them with T7 RNA polymerase and labelled NTP
What is end labelling?
Incorporates only a single label - GAMMA phosphate from ATP is transferred to DNA by polynucleotide kinase - (T4)
Useful for tracking the ends of DNA but no so useful for labelling longer DNA molecules in some restriction experiments because only one label is incorporated per probe strand.
What happens with oligonucleotides?
T4 polynucleotide kinase adds gamma phosphate
More than one label per oligo - can be used in high concentrations
Describe the uses of digoxigenin?
DNA probe that undergoes nick translation or end filling with digoxigenin-UTP. Hybridise the probe and detect with anti-DIG antibody coupled to a fluorescent marker or enzyme conjugate.
Secondary antibody = HRP or alkaline phosphatase
What do you detect biotin with?
Avidin
What is a microarray experiment?
Analysis of expression of thousands of genes in a single reaction
Hybridise mRNA to the DNA for the corresponding genes
DNA immobilised on microarray
Detect the amount of mRNA that binds through fluorescence
What is the MMTV promoter system induced by?
Glucocorticoid
What do mammalian expression vectors express for propagation in E.coli?
Beta-lactamase and origin of replication
What does neomycin resistance show?
Selection in cultured mammalian cells
What is tet-on?
TRE bind the trans-activator protein with tetracycline-like drug is present (doxycycline)
What is tet-off?
TRE binds trans-activator protein when tetracycline-like drug is not present.
Binding of doxycycline to the trans-activator protein causes it to no longer bind to the TRE
What does the text system require?
Two plasmids - one with TRE gene and one with trans-activator
What is the natural life cycle of the baculovirus?
Caterpillar ingests viral particles and then alkaline pH in the gut causes the particle to dissolve - releasing the viral capsids
Taken up by gut epithelial cells - replicate and assemble by commandeering the cells own transcription/translation machinery
Multiple capsids are packaged into polyhedron coats and eventually leads to cell death and lysis of the cell
How is the gene of interest transformed into the bac vector?
Gene of interest is cloned into a transfer vector - recombines with baculovirus DNA to produce recombinant virus DNA - LacZ shows successful recombinants.
What is the expression system in baculovirus particles?
Recover high molecular mass backed DNA from E.coli and check for insert by PCR
Transfect insect cells and recover budded virus
Transfect cells as required for expression
Constitutive expression from viral polyhedron promoter
Protein can be secreted by making fusion with BiP or honey bee melting leader sequence
What is the recognition sequence for viral RNA genome?
Psi sequences
What are the two pathways of RNA degradation?
Decapping (deadenylation-independent pathway) and deadenylation dependent pathway
Also endonucleolytic pathway
What triggers sequence specific down-regulation of protein expression?
dsRNA - Potent and specific interference
What are the two RNAi pathways?
RISC - recognises specific sites and cleaves the target - small dsRNA pieces are used for targeting complementary sequences within mRNA
Translation repression by targeting mRNA into P-bodies - involves microRNA
What is the pathway of microRNA?
Produced as sRNA by Pol II as tandem copies
Fold into sRNA hairpin
Processes in the nucleus by Drosha/Pasha (Exportin-5)
Exported to cytoplasm and processed by DICER
What are the two types of interfering RNA?
microRNAs - most come from RNAs transcribed in the nucleus and then fold into hairpins
small interfering RNAs - derived from dsRNA
What ribonuclease III is at the heart of DICER?
Argonaut - catalyses cleavage of mRNA
Double stranded precursors are cute by what?
DICER - 21 nts
The binds Argonaut - one strand remains bound (RISC formation)
siRNA direct RISC to bind to specific mRNA - targeting is precise determined by watson crick
What do microRNAs do?
Direct RISC to mRNA - imprecise so can target 100s of endogenous RNAs - can lead to mRNA degradation or blocking of translation
What are the purposes of the silencing pathway?
Temporary knockout - functional genomics in cell culture
- Functions of genes can be mapped
- Screening for drug targets
RNAi based therapeutics
What are the stages of turning sRNA into a drug?
Sequence selection
Synthesis and modification
Packaging and delivery
Targeting
What could cause differential gene expression?
Gene loss
Gene amplification
Different expression
What experiments showed genes are not lost?
Frog skin cell
Carrot cell
What is the only bit of evidence for gene amplification?
Chorion gene - encodes protein needed for egg shells
Ribosomal RNA genes - found in Xenopus eggs - oocytes have a huge number of ribosomes therefore rRNA genes are amplified
How is gene expression studied?
Southern blot In situ hybridisation Quantitative PCR Microarrays Replace ORF with a reporter protein
Where is the EVE gene found?
Fruit fly - defines body segment formation
What is eve expressed as?
7 stripes
What is the eve gene regulated by?
Long upstream regions, 7.3kb
Switches eve on - defines where eve is expressed - expression of eve in each of these 7 stripes is controlled by independent sequences in this region
What is the strategy to define the sequence?
1) Substitute eve ORF for a reporter gene
2) Make deletions to the 7.3kb regulatory region
3) Introduce these altered genes into drosophilia eggs
4) Does gene expression still occur?
What restriction enzymes removed stripe 2?
BstE11 to BssHII
What does ExoIII digest?
3- termini of an expose duplex DNA
What does Bal31 digest?
Digests both strands of an exposed DNA duplex
What are the four regulatory proteins in eve?
Kruppel (-)
Hunchback (+)
Bicoid (+)
Giant (-)
What are the four main methods of studying the regulatory proteins?
Gel shift mobility assay - see how far the DNA and protein run (move less than just DNA)
DNA affinity chromatography - Add DNA to solid matrix and bind to a column then add cell lysate
DNA footprint analysis - Cut with Dnase1 - protein protects
Chromatin immuno-precipitation
How do you insert a transgene into all cells of an organism?
1) Introduce plasmids by injection into G0 embryo
2) Transposon integrates into nuclei at embryo pole
3) Pole nuclei become pole cells - these form germ line
How do you do gene transfer in plants?
Uses a bacterium - Agrobacterium tumeficiens
Naturally infects plants
Can transfer its own DNA to plant cells during infection - mediated by Ti plasmid (dependent on T-DNA repeats)
What is the process of gene transfer into plants?
1) T-DNA repeats are excised and passes into the plant
2) Infect plant cell cultures
3) Cultured in nutrient media
4) Antibiotics used to allow selection of transgene recipients
5) Only transgenic plants survive
What are the main aims of generating a transgenic organism?
1) Understand function (cells in culture)
2) Aspects relating to differential expression (intact organism)
