What does Sanger Sequencing utilise to create differently sized nucleotide strands?
dATPs are used for chain extension
Fluorescently labelled ddATPs are used as terminator nucleotides as they don’t have an OH on the 3’ end
How were DNA fragments cloned in second generation sequencing?
100-200bp fragments cloned into bacterial artificial chromosomes (BACs)
What advantages did next generation sequencing have over sanger sequencing?
Lots of molecules could be sequenced at a time
Price per base dropped drastically
Fluorescent bases used
Bridge amplification for detecting the template molecule
What sort of cloning did massively parallel sequencing use?
Bridge amplification
1. DNA fragments isothermally amplified
2. Oligos are added which hybridise with the lawn adaptor region
3. A complimentary strand is amplified through bridge amplification
4. Chain extension is prevented by reversible terminator nucleotides
5. The colour that fluoresces corresponds to the nucleotide
What advantages do third gen sequencing have over illumina?
Single molecule sequencing
Real time sequencing
Ultra long read lengths
Identifying base modifications
What disadvantages do third gen sequencing have over illumina?
More expensive per base
Higher error rate
Outline PacBio sequencing
- DNA is ligated into SMRT-bell adaptors
- These are put into zero-mode waveguides (ZMWs)
- Fluorescently labelled primers attach to the adaptors and go round the entire DNA, giving a circular consensus sequence
- Fluorescence is detected from a single base and gives a pulse corresponding to the nucleotide
Outline the GridIon machine mechanism
- Motor protein unravels DNA and passes it through the pore into solution
- Another molecule can the attach and give direct sequencing of DNA/RNA/protein
How can the level of expression of a genome be quantified?
RT-qPCR:
1. RNA has cDNA synthesised from it with reverse transcriptase
2. DNA is amplified via PCR
How can the level of expression of a genome be quantified?
RT-qPCR:
1. RNA has cDNA synthesised from it with reverse transcriptase
2. DNA is amplified via PCR
What can be used to determine the genes being expressed?
Northern blotting:
1. mRNA is separated with electrophoresis
2. purified mRNA is transferred to a membrane
3. A labelled probe is used to find the transcript
(this is not very quantitative)
What are some limitations of microarrays?
Low resolution sequencing as the probe may contain a similar sequence
Have to be predesigned, so we don’t know if any probes are missing
Not very quantitative as there is a limit to the amount of RNA that can hybridise in one spot
How is DNA methylation on a genome found?
Bisulphate sequencing.
Sodium bisulphate converts unmethylated cytosine to uracil
How is DNA fragmented for bisulfate conversion?
DNA is digested with a restriction enzyme MSP1 which recognises CCG
How are DNA binding proteins bound to DNA detected?
Chromatin Immunoprecipitation:
1. Proteins are covalently cross-linked to DNA by formaldehyde
2. Chromatin is sheared by sonication or endonucleases. ChIP-exo trims DNA to the binding site
3. Bound DNA is purified and immunoprecipitated with specific antibodies, then put in a microarray
How are RNA binding proteins bound to RNA detected?
CLIP-seq
RIP-seq
What is used to identify long range chromosomal interactions?
3C: One enhancer to an interacting region
4C: One enhancer to al its interacting regions
5C: Many enhancers to many tagerts
Hi-C: All enhancers to all interacting targets
How are essential genes found?
TraDIS:
Genes are inactivated with transposons
Chromosomal regions are sequenced outwards from inverted repeats
What is used to find transcribed regions?
RNA-seq
How are open chromatids found?
DNAse-seq and FAIRE-seq
How are DNA satellites identified?
DNA fingerprinting:
1. Northern blot DNA
2. Minisatellite sequences are used as a probe
3. Two minisatellite sequences for each probe show up
How are animal origins of replication found?
DNA is treated with BrdU (thymidine analogue)
Microarrays are used to find where origins of replication are
How is amount of protein expressed and by how many cells found?
GFP promotor trap:
1. Genes from promotor libraries are plated
2. FACS separates cells by active/inactive promotor
3. Inactive promotor cells are put into the animal model and the active cells taken out are quantified
Who is phenotypic screening done?
BioLog array
