Genes, Chromosomes Flashcards
What are ddATP nucleotides and what is their role in Sanger Sequencing?
ddATPs are terminator nucleotides that halt chain extension; markers on these nucleotides allow for generation of a sequence.
How does the structure of ddATP nucleotides differ from dATP?
ddATP doesn’t have an OH group attached to the deoxyribose ring, preventing further chain extension.
What are Sanger’s two greatest accolades?
2 nobel prizes:
1958 - structure of proteins and insulin
1977 - sequencing nucleic acid (sanger sequencing)
How is sanger sequencing performed in a singular reaction?
Utilising fluorescently labelled ddNTPs, allows for the process to occur in a single capillary tube; these are then separated by length during electrophoresis.
Give an example of a device that can electronically read DNA sequences by Sanger Sequencing:
ABI 3730 sequencer -> can sequence up to 800bp from a single reaction
How long is the human genome?
3 billion bp across 24 chromosomes.
What was the human genome project’s approach to sequencing the genome?
Extract the human genome DNA anonymously -> fragment the DNA via sonication -> select by size (via gel electrophoresis) -> 100-200kb fragments were cloned in BACs -> E.coli then used to amplify DNA.
What are BACs (Human genome project)
Bacterial Artificial Chromosomes (pretty much plasmids)
What is the loop of BAC clone by clone method? (human genome project)
Start with a genetic map marker -> perform PCR on known BAC library -> Identify BAC clone with genetic marker ->[ PCR using primers based on seq. from ened of BAC insert -> add to BAC library -> identify the clone containing the end seq ->] repeat.
Summarise how BAC clone by clone method worked? (Human genome project)
The genome is slowly identified by the overlapping sequence of clones, which span the difference between markers on the genetic map.
What is a genetic map?
A genetic map shows the relative location of markers on a genome.
Shotgun Approach:
Celera
40-100Kb DNA fragment from BAC clone -> broken into 5-10kb fragments -> inserted into vectors -> 800bp chunks sequenced from either end -> generates pairs of sequences that can be pieced together computationally.
What had shotgun sequencing been used for before the human genome project?
The sequencing of Haemophilus Influenzae genome.
Why didn’t the human genome use shotgun sequencing?
Government funders are risk adverse.
Assembly and closing gaps was easier using clone by clone.
What was the name of the private company that competed with human genome project?
Celera
When was the human genome published?
February 15th 2001
What strategy is commonly used in modern sequencing?
Shotgun Strategy.
Original idea/method of Sanger sequencing:
To produce many fragments of DNA of varying sizes by performing PCR with free nucleotides and a specific ddATP. gel electrophoresis is then performed to identify the positions of the base attached to the ddATP.; this is repeated four times each time with different base.
What machine uses massively parallel sequencing?
Illumina’s HiSeq.
How many reads can be produced from one HiSeq run?
8 billion sequence reads -> each with a max length of 125bp.
What type of amplification does illumina sequencing employ to amplify the template molecule?
Bridge amplification (illumina occurs on surface rather than in solution)
Why does illumina need to amplify the template molecule:
The optical sensors aren’t sensitive enough to detect a signal from a single molecule.
What are the keystages of illumina?
-Genomic DNA extraction
-DNA fragmentation
and size selection
-Library preparation (addition of adapter molecules)
-Cluster generation (bridge amplification)
-Sequencing by synthesis
-Data analysis
What size fragments are used in illumina sequencing?
500bp
What is attached to DNA fragments in illumina sequencing?
adaptor sequences on the 5’ and 3’ end. This allows for the hybridisation of primer sequences (base pairing) -> allowing for amplification and formation of new strand.
How does bridge amplification work?
The template strand binds to one of the fragment strands on their adaptor region. A polymerase creates a complement of the hybridised fragment, the molecule is denatured and the template is washed away. The strand folds over and binds to a complimentary adaptor seq. A polymerase forms a complement. The molecule denatures however both strands are fixed to the surface and-so can repeat the process with other adaptor strands.
How do clusters form in illumina sequencing?
The template sequence fragments attach to the flow cell surface at random positions, undergo bridge amplification, forming clusters.
How can separate clusters be formed from one flow cell?
By controlling the concentration of template fragments across the cell.
What method underpins the approach of illumina sequencing?
Shot gun sequencing -> many random clusters simultaneously sequences and analysed to build sequence,
What approach does illumina use to build-up a DNA sequence?
USes a sequencing-by-synthesis approach.
What is a sequencing-by-synthesis approach?
Uses fluorescently labelled reversible terminator nucleotides which can then be cleaved and further extended. Extension process can be controlled chemically.
What enzyme breaks down DNA strands, strand specifically?
Endonuclease
In which direction does sequencing occur in illumina sequencing?
Common for it to occur in both directions
How far apart are reads sequenced from the same fragment in illumina sequencing?
500bp apart.
What is purpose of adding a 3rd primer in illumina sequencing?
For index sequencing:
Allows for different samples to be distinguished. -> maximising the data generated by the machine.
What generation of sequencing is illumina?
Next gen Illumina
What is an example of next gen sequencing?
Illumina
How many primers are needed for bridge amplification?
2 primers
What are the advantages of Second Gen Seq?
-Massively Parallel
-Much cheaper to sequence per base
“built-in” shotgun sequencing without cloning step
What are the disadvantages of Second Gen Seq?
-Library Prep can be expensive and slow
-Amplification of DNA fragments is required which can introduce bias against CG sequences
-Read lengths are short and smaller than sanger
What are the advantages of third gen seq?
- Single molecule sequencing (no amplification) -> no bias
-Real time sequences (generated during run)
-Ultra-long read of up to 50kb (PacBio) or >2Mb(nanopore)
-Can identify base modifications
Disadvantages of third Gen seq:
-Fewer reads per run. (more expensive per base)
-Individual reads have high error rate - (Caused by no redundancy because it’s a single molecule.)
How can the high error rate of third gen sequencing be circumvented?
By performing multiple reads to retrieve a consensus read.
What are the key features of PacBio seq?
-Sequence-by-synthesis
-Fluorescently labelled nucleotides
-Single molecule at a time
-No pause required between base incorporations
-Zero-mode waveguides
-Individual reads have high error rates.
How are the high error rates of PacBio Seq compensated?
Circular consensus seq.
What is the role of zero-mode waveguides?
To allow for the detection of fluorescence at a single molecule level.
What is a key distinction between marker nucleotides used in third gen vs second and first gen?
The nucleotides aren’t terminator nucleotides, and instead the fluorescent label is cleaved.
Why don’t most the free nucleotides fluoresce in PacBio seq?
The illuminated region is very small surrounding the template fragment, and therefore there’s only small amounts of background noise from some diffusing into the illuminated space.
What is the structure of a PacBio SMRT chip?
The sequences on the surface of 150,000 ZMW wells, each well has an immobilised polymerase.
What is the process of sequencing in PacBio SMRT chips?
Replicate template DNA molecule, incorporates fluorescent nucleotide into single DNA strand -> label is cleaved as the polymerase moves along the sequence -> sequence extension.
What is the process of circular consensus seq?
-Double stranded DNA -> SMRT-bell adaptors added -> creates closed loop of DNA -> Primer used to hybridise with adaptor seq -> polymerase replicates the fragment. -> at junction the polymerase displace the second strand, allowing for replication of the lower strand -> this process loops -> the subreads can then be sequences and a consensus sequence can made.
What are the components of Oxford nanopore?
-An artificial electrically insulating membrane
-Motor Protein (polymerase or helicase)
-CsgG pore protein
What is the error rate of Oxford Nanopore?
~1%
What can Oxford nanopore sequence?
DNA, RNA, and proteins.
How does oxford nanopore work?
As each nucleotide/component crosses the membrane through the CsgG pore the shift in electric current is used to deduce its identity sequentially.
What happens to the motor protein in oxford nanopore once sequencing has finished?
the motor protein will fall off.
Why are repetitive regions an issue for shotgun sequencing?
If they’re large relative to the size of fragment or reads they can be difficult to locate.
What does software do if it can’t form a continuous sequence due to repeat regions?
It will form contigs/draft sequences.
What is the process of resequencing?
Mapping to a reference genome, taking sequence reads, working out where they derive from the reference, differences can be compared.
What is meant by functional genomics?
Methods to understand genome function by mapping regions of the genome to reference.
What are examples of functional genomic methods?
RNAseq + Chip Seq.
Genomics:
The structure of the genome.
Transcriptomics:
Which genes are when + how much are they expressed.
How can the genomics of a biological sample be investigated?
Either the generation of a consensus seq. via DNA sequencing or Resequencing from a reference genome
Steps of Northern Blotting:
RNA extraction from sample -> electrophoresis (separate by size) -> northern blotting (Transfer of RNA to Membrane) -> tag RNA using radiolabelled probes -> visualise under x-ray
What is RT-qPCR?
Reverse Transcriptase quantitative PCR is a method to investigate Transcriptomics.
What is the process of RT-qPCR?
One gene at a time: RNA reverse transcribed into cDNA -> undergoes PCR amplification -> relative amounts of fluorescent primers will give information on relative expression of different regions at different times.
How are microarrays used for transcriptomics?
Spots loaded with different sequences -> fluorescently tagged mRNA nucleotides bind to complementary bases in samples -> relative fluorescence of each sample gives level of expression.
What are technical replicates?
Same procedure with same sample -> variation caused by equipment
What are biological replicates?
Independent experiments with cells from different cultures.
Transcriptomic experiment output equation:
Log2(full change)
What is the transcriptomic experiment output?
A measurement of the effect size of variation.
How can RNA seq. be used for transcriptomics?
RNA is fragmented -> reverse transcribed into cDNA -> sequenced. The reads are then mapped and genes that match more of the reads than other are more expressed.
Limitations of MicroArrays:
-Low resolution
-Exact sequence present unknown
-No info on sequences not covered by probes
-Limit to how much RNA can hybridise to a particular spot -> limits ability to distinguish expression at higher levels.
RNA seq vs Micro Arrays: Transcriptomics
- Both well developed with minimal technical variation
- RNA-seq has larger more dynamic range + can distinguish levels of expression more clearly
-MicroArrays only give info on preselected region
-RNA-seq is genome wide
-RNA-seq allows us to detect differences from the ref genome
Alternative splicing:
1 pre-mRNA -> multiple alternate spliced forms -> multiple protein isoforms
What is DNA methylation?
A method of modifiying/regulating gene expression wherein a methyl group is added to the 5 carbon of the cytosine base -> this down regulates gene exrpession.
Examples of DNA methylation:
-When genes are silences - e.g. X inactivation
-Imprinting: distinguishing maternal and parental alleles.
-In bacteria to distinguish between “self” DNA and foreign DNA.
What is a CpG site?
A site where a C is connected to G by a phosphate group rather than base paring.
What is the role of CpG islands in gene supression?
CpG islands are sometimes within promoter regions. methylation of cytosine groups will cause the gene to be repressed, however this will then spontaneously deaminate into thymine, permanently silencing the gene.
What is Bisulphite Conversion a method used for?
To investigate the methylation of DNA,
How does Bisulphite Conversion work?
DNA is treated with sodium bisulphites -> incubated -> unmethylated cytosine will convert into uracil and-so remaining cytosine bases are methylated.
What is RRBS?
Reduced Representation Bisulphite Sequencing.
What is A-tailing?
An enzymatic method used to add a non-templated nucleotide to the 3’ blunt end of a double stranded DNA molecule.
Steps of RRBS (Reduced Representation Bisulphite Seq.)
Purify genomic DNA -> restriction enzyme digest (with CpG recognition site) -> A-tailing -> adapter ligation -> gel electrophoresis (size selection for smaller fragments)-> bisulphite conversion -> PCR amplification -> sequence.
How can a methylated base be identified by PacBio seq?
They will be a longer pause between two signals.
What is Chromatin Immunoprecipitation?
A method used to isolate DNA bound by specific proteins.
In Chromatin Immunoprecipitation, how is the DNA crosslinked to protein>
Protein-DNA crosslinks are formed using formaldehyde.
Steps of ChIP (Chromatin Immunoprecipitation)
DNA and protein treated with formaldehyde -> chromatin sheared by sonication or endonuclease (get rid of protein unbound DNA) -> use of exonuclease allows for DNA to be trimmed to the binding site -> Antibody specific to the protein of interest is bound to a membrane/matrix and used to purify and immunoprecipitate the protein and its bound DNA.
How can the DNA purified by ChIP be analysed?
The DNA can be purified and separated from the bound protein and then either hybridised to a microarray, or fluorescently tagged
What is ChIP-seq?
A method using Illumina sequencing to characterise the binding sites of a protein.
What is the process of ChIP-seq?
Purify DNA from bound protein -> Digest DNA upstream of the protein using 5’ to 3’ exonucleases. -> map sequences to reference -> both strands will have a small over hang/overlap on reference map -> this is the region the protein binds.
What is Chromosomal Conformation Capture?
A method of observing long range chromosomal interactions. (E.g. sequences/components very far apart in the sequence but close in proximity)
Give an example of a long range chromosomal interaction:
Enhancers -> similar to promoters they promote the expression of a specific gene -> however unlike promoters they are very far away in the sequence, but close by physical proximity.
What are the different types of Chromosomal Conformation Capture?
3C, 4C, 5C, and Hi-C
3C Chromosomal Conformation Capture: Outcome
3C assesses the presence of interactions between known sequences.
How does Chromosomal Conformation Capture work?
It looks at cross-links between “remote regions” of DNA -> the DNA is digested using a restriction digest (fragmented) -> the ends of the fragment are ligated to form closed “chimeric DNA” -> the cross-link is removed -> either purified and amplified by PCR or sequenced.
4C Chromosomal Conformation Capture: Outcome
Allows for the assessment of one known sequence with multiple others.
5C Chromosomal Conformation Capture: Outcome
Allows for many different interactions within one region to be observed.
Hi-C Chromosomal Conformation Capture: Outcome
Allows for the observation of every long-range interaction within the genome
3C Chromosomal Conformation Capture: How does it work
PCR with primers specific to both interacting regions
4C Chromosomal Conformation Capture: How does it work
Digestion with 4bp by restriction enzyme -> ligate to form circular DNA -> amplify by PCR with primers from one region-> Microarray
5C Chromosomal Conformation Capture: How does it work
Design lots of primers for many potential interacting partners -> ligation and PCR -> Microarray/sequening
Hi-C Chromosomal Conformation Capture: How does it work
Biotin is used to label junction -> This complex is sheared -> ligation -> travertine recognises the biotin and purifies the DNA -> adaptors are ligated + amplification _> sequencing
5C Chromosomal Conformation Capture: Primers
Each primer has a universal tail, meaning once they hybridise they can be amplified.
What is ENCODE:
The Encyclopaedia of DNA elements.
What were the techniques employed by ENCODE to find functional genes?
-5C Chromosomal Conformation Capture -> ChIA-PET
-DNase-seq and FAIRE-seq
-WGBS (whole genome bisulfide seq.)
-PCR
-RNA-seq
-CLIP-seq + RIP-seq
Role of CLIP-seq and RIP-seq in ENCODE:
TO identify proteins crosslinked to RNA
What was the role of DNase-seq and FAIRE-seq in ENCODE?
To look for open chromatin
DNase-seq: How does it work?
Exploits that when DNA is unwound it’s accessible for degradation
FAIRE-seq: How does it work?
Crosslinks histones to DNA and identifies regions that are too tightly wound to be bound to proteins
Evolutionary Conservation as an indication of function:
-Histograms can be used to compare relation between organisms.
-Exons are more highly conserved between species -> due to mutations in exons likely being negative for viability
What are transposons?
DNA sequences that can more from one genetic element to another, containing genes additional to those require for transposition.
What is TraDIS?
Transposon Directed Insertion-site Sequencing
Structure of a transposon:
Short DNA sequence with a transposase gene flanked by two regions of inverted repeats.
What is the role of the inverted repeats on a transposon?
They are recognised by transposase and allow for the transposon to then move and integrate elsewhere.
How does TraDIS operate?
Millions of bacterial mutants are produced by inserting an engineered transposon into the cell -> genes are disrupted when a transposon inserts into them -> sequence reads are taken of each mutant -> reads are mapped to reference genome -> regions of no noise are essential.
What would a change in TraDIS results between environments suggest?
That specific genes are more/less essential to the fitness of the bacterium dependent on the host/environment.
What are the advantages of using microbes in genetics?
-Simple to maintain
-Reproduce Rapidly
-Spontaneous mutations will occur in large populations
-Easy to select
-Haploid -> phenotype of mutation is immediate
-Small genome size
What are known as conditional lethal mutations?
Mis-sense mutations that cause changes to protein structure/function in specific conditions (e.g. high temperature or pH)
What is an alternative name for a point mutation?
A single nucleotide polymorphism.
What causes frame shift?
Insertion or deletion of a single base pair, causing changes in the bases read by the ribosome, altering all codons downstream of the mutation.
What are potential causes of spontaneous mutations in microbes?
-Deamination by water
-EM radiation
-Analogue Bases
- Base Modifying Chemicals
-Transposons
-Recombination
-Lambda Red
-Intercalator insertion
Error Proof DNA repair:
There are many repair systems, each recognising a specific type of damage.
Hemi-methylated DNA:
DNA that is methylated on the parent strand, but not the newly synthesised strand.
What does the MUT system do?
The MUT system repairs mis-matched regions that don’t base pair on Hemi-methylated DNA.
How does the MUT system work?
Mut S binds to the region where base pairing does not occur -> this recruits MutL and MutH -> MutL brings its domains together to form a loop with the damaged DNA -> MutH acts as an endonuclease and creates a nick -> UvrD unwinds the two strands -> an exonuclease will then cleave the erroneous DNA -> DNA polymerase 1 then ligates this gap
What is a thymine dimer?
Where two adjacent thymine bases on the same DNA strand base pair with eachother.
What does Nucleotide Excision Repair Do?
Repairs thymine dimers induced by UV damage.
How does Nucleotide Excision Repair work?
UvrA and UvrB recognise the thymine dimer -> they bind to it and bend the DNA -> This is then recognised by UvrC (endonuclease) -> this creates a nick either side of the dimer -> helicase then unwinds the DNA and erroneous portion is removed -> DNA polymerase 1 fills and ligates the gap.
What does base excision repair fix?
Portions of DNA where the base has been damaged.
How does base excision repair function?
Base excision repair -> DNA glycosylate removes the damaged base leaving an “AP” site-> Ap endonuclease creates nick by AP site -> UvrD helicase unwinds the DNA -> DNA Pol1 fills in the base -> ligase then ligates the strands together.
What does recombinational repair fix?
Gaps in strands formed during strand replication. e.g. caused by replication of strand with thymine dimer.
Slip-strand mis-pairing(slippage):
Tends to occur in sets of three bases in large repeat sequences -> the strand will slip back, causing the addition of an amino acid.
What is the term given to the bacterial use of slippage in control of gene expression?
Phase shift.
How does recombinational repair work?
RecA aligns homologous regions in the DNA -> finds region homologous to the gap -> strand invasion occur -> sister strand loses region of strand to fill in gap -> DNA polymerase 1 will fill in the gap of the sister strand.
What does the SOS repair system fix?
Used when there is extensive DNA damage across the chromosome.
How does the SOS repair system work?
Once activated, SulA (stops cell div), UmuDC - (encodes DNA Pol V), and UrvA (NER pathway) are expressed.
How is the SOS system regulated?
The system is kept of when the genome is healthy, by using a LexA repressor. When DNA is damaged ssDNA builds up -> this is recognised by RecA which will then repress LexA -> activating the SOS sytsem.
What is DNA Pol V not used ordinarily despite it’s fast speed?
It has a high error rate.
What is mutation frequency?
The number of mutants within a population.
What is the mutation rate?
The estimation of the probability of a mutation occurring per cell division.
What is the difference between bacterial selection and screening?
Selection uses growth media that will only grow the mutant whereas screening both the wild type and mutant can grow however can be phenotypically distinguished.
Site-specific Recombination:
The phage DNA has an att site (attP) -> integrase helps the DNA integrate at the attB on the chromosome.
Homologous Recombination:
When two pieces of DNA with identical sequence recombine.
When does Homologous recombination occur?
When linear of plasmid DNA is unable to replicate but has homology with the chromosome between 500-1000Bp.
What is horizontal gene transfer?
Mechanism whereby genes can be spread to different cells and species -> occurs when a piece of DNA is taken into a cell.
What are the two functions of Recombination?
Horizontal Gene Transfer and Recombinational Repair.
What is the Chi-site?
A nucleotide sequence which stimulates the Rec-BCD pathway in homologous recombination.
How does strand invasion in homologous recombination occur?
RecB, RecC, and RecD enter at the end of the Donor DNA fragment and unwind it -> this occurs until the chi site -> the DNA is nicked and further unwound -> forms an ssDNA arm. RecA binds to the ssDNA and aligns it to a region of homology on the chromosome. Strand invasion occurs.
What structure forms during strand invasion during homologous recombination?
A D-loop (single strand crossover)
What occurs after strand invasion during homologous recombination?
RuvA and RuvB assemble at the single strand crossover and pull the donor and recipient strands in opposite directions (branch migration), an endonuclease cleave the end of the D-loop and the strands are ligated together. This leaves one crossover known as the “Holliday Junction”
How many crossovers are present in a D loop (single strand crossover?)
2 crossovers.
What are the two possible outcomes of cleaving the Holliday junction?
There will either be two identical strands of DNA or the two strands will vary with one being sandwiched by donor DNA and one sandwiched by recipient DNA.
What happens in homologous recombination is the donor DNA is plasmid DNA?
A co-integrate will form -> the two will merge as a singular product.
What occurs in homologous recombination if the donor DNA is linear?
A 2nd crossover event must occur to maintain the circularity of the bacterial genome. Some of the DNA from the linear will have been incorporated into the chromosome. However the leftover linear molecule is degraded by nucleases.
Summarise homologous recombination:
New DNA fragment introduced -> will be digested by nucleases unless it shares region of homology with host chromosome -> Linear DNA processed by RecBCD -> RecA binds to the ssDNA strand and aligns it to region of homology on chromosome -> Intermolecular recombination by Ruv proteins leads to formation of D-loop -> crossover is cleaved, followed by Holliday junction.
What enzyme cleaves the Holliday junction?
RuvC
What are the 3 primary components of lambda red?
Exo, Beta, and Gam.
Lambda Red: Exo
Exo is a 5’ to 3’ exonuclease that degrades the 5’ ends of linear DNA to form ssDNA.
Lambda Red: Beta
Beta binds to ssDNA at 3’ end and promotes annealing to complementary DNA. (similar to RecA)
Lambda Red: Gam
Binds to the host RecBCD system to inhibit its activity.
What is Lambda?
A bacteriophage that has its own recombination system.
How do we use homologous recombination to make knockout genes in labs:
Host Chromosome has 3 neighbouring genes A,B, and C. To replace B with b -> construct PCR product with 1000 base pairs homologous to Genes A and C, in between b gene with kanamycin resistant antibiotic cassette -> double recombination event occurs -> gene B will be replaced and linear bi-product is digested. -> can select for mutant using kanamycin plates.
Why is it beneficial to co-opt the lambda red system?
Lambda Red is a much better and faster method of gene knockout in the lab.
How are protein components of the lambda red system introduced into badcteria?
A plasmid delivery system is used.
Specifically plasmid PKD46 -> which has a temp sensitive origin of replication.
How do PKD46 operate in lambda red gene knockout?
It operates as a plasmid delivery system. The plasmid has a temperature sensitive origin of replication. At normal growth temperature the proteins are expressed (at 42’C they aren’t). Each protein gene is contained within the same operon controlled by the pBAD promoter (activated by AraC TF detecting arabinose).
Why is the temperature sensitive nature of PKD46 important in lambda red gene knockout?
The plasmid can be controlled to prevent unwanted expression. -> once expression is halted the plasmid will be broken down by nucleases.
How long does the lambda red gene knockout take for E.coli?
One week.
How can the lambda red gene knock out be improved?
By limiting the exposure of the cell to labra red activities and include sacB based counter-selection.
What is sacB counterselection:
sacB is a gene from Bacillus Subtylus -> uses sucrose to make levans -> toxic to -ve bacteria .
What is counterselection?
Genetic engineering techniques used to eliminate specific gene fragment’s containing selectable markers.
Example of Lambda Red more efficient method:
-Lambda red now also includes mega nuclease SceI and has a SceI cleavage site (cassettehas two cleavage sites)-> cleavage causes formation of linear donor DNA -> increases the amount of homologous DNA to the genome + sucrose to ensure leftover plasmid stops functioning -> select using kanamycin to select for successful mutants.
What are the two groups of transposable elements?
Insertion Sequences + Transposons
What are insertion sequences?
DNA sequences that can travel around the DNA but only code for their transposition.
Transposable Element Characteristics:
-Infrequently move (10^-7 per generation)
-Don’t require homology to DNA seq.
-Utilise Transposase
-Ubiquitous
In which type of organisms does Transposon Mutagenesis function?
Bacteria because they are genetically tractable.
Structure of insertion sequences:
1-2kb, contains DNA encoding for the transposase gene flanked by terminal inverted repeats.
What are the two types of transposons:
Complex Transposons and Composite Transposons.
Composite transposon:
-Interior inverted repeats
-Additional genes flanked by degenerated inverted repeats insertion sequences on either end of the transposon.
-Unable to replicate
Complex transposon:
-encodes for transposase, resolvase, and more.
-All flanked by degenerated inverted repeats.
-Replicative
Replicative Transposition:
Transposable element is copied, with one remaining within original site.
Non-Replicative Transposition:
Transposable element jumps between sites.
What is the process of non-replicative transposition?
Transposase aligns the inverted repeats by folding the DNA -> cleaves phosphodiester bond on both strands at both side of the IS element-> 3’-OH’ attack produces hairpin structure and host carrier DNA is ejected and repaired. -> hairpins are re-nicked and 3’-OH attack recipient DNA -> transposasome move to target DNA seq -> insertion.
What kind of cut is made to DNA by transposase?
A staggered cut to allow for ligation.
What is a transposasome?
The complex of transposon and transposase.
How do transposons change the genome even if they leave a site again?
Every jump creates duplicated regions created by transposase’s staggered cuts.
What are polar effects:
A change to the genome that causes downstream effects to other genes (e.g. a transposon inserting in the first gene of an operon)
What is the effect of a transposon inserting into the gene on its function?
They will inactivate a gene, by causing a frame shift/ null mutation.
What is TMDH?
Transposon-mediated differential hybridisation.
What is TMDH a precursor of?
TraDIS
What is the specific about the modified transposon used in TMDH?
Instead of inverted repeats the transposons have a T7 promoter on the 5’ end and SP6 promoter on the 3’ end.
What was the goal of TMDH?
To generate a library of mutants
Process of TMDH:
Insert modified transposon (Tn) into Bacterium -> culture -> extract genomic DNA and fragment by restriction endonuclease. -> transcription in vivo using a polymerase for each promoter region on the Tn and marked dNTPs-> generate run-offs -> hybridise to whole genome microarray.
Does the lambda red system also use the chi site?
No
What is RNAP?
RNA polymerase/
Where does RNAP bind to a gene?
Upstream of the gene’s promoter region.
Transcription Fusions:
A transcript is engineered with the promoter of a test gene and the translational elements of a reporter/marker gene to observe changes to the expression of the test gene. -> forms two proteins
Translational Fusions:
The promoter and translational elements of the test gene are used to drive the expression of the reporter -> therefore allows for the determination of gene expression in different environments. -> forms one chimeric protein
lacZ: function in lab
Encodes Beta-galatcosidase -> can observed using colourimetric assay
cat: function in lab
Encodes chloramphenicol acetyltransferase -> will be resistant to chloramphenicol (screening)
lux: function in lab
Encodes luciferase -> can be quantitatively measured -> needs O2 to function
gfp: function in lab
encodes green fluorescent protein -> allows for cell imaging of protein expression and localisation
What does a difference in the expression between transcriptional and translational fusion confirm?
That post-translational transformations are occuring.
What are single copy reporter fusions used for?
A method to investigate the transcriptional and translational expression of gene.
What is FACS?
Fluorescence Activated Cell Sorting -> to separate clones that are already activated outside the host -> isolate not-activating genes.
GFP promoter trap: what is this method kind of similar to?
TraDIS combined with innoculation.
GFP promoter trap: Process
Digest bacterial DNA and clone into promoter-less GFP plasmids -> isolate unactivated clones by FACs -> innocculated into host-> bacteria is recovered and isolate activated clones by FACs -> this indicates which genes are activated in vivo.
