Next generation sequencing and micro-arrays Flashcards
How are microarrays performed?
Take 2 populations of DNA:
Add different fluorescent tags to the different populations. Then mix the DNA populations and put them into the wells of a chip. Each well has a single stranded oligo tethered to it. This oligo might be WT or DNA containing a specific mutation. If the normal or tumour DNA binds to an oligo then that fluorescent tag will remain in the well after washing. This can be interrogated through fluorescent microscopy.
What would be the result in a well comparing tumour Vs WT DNA where oligos are fishing for specific somatic mutations
Tumour DNA will be bound in wells that contain the same mutation and the colour of the fluorophore that was bound to the tumour DNA will be present.
What is the result in a well that is fishing for mutant DNA when comparing tumour DNA and normal DNA that contains a germline mutation
Both sets of DNA will hybridise so a mix of the colours will be seen in the well.
What is required when performing a micro-array
Need to know what you’re looking for so oligos can be designed for certain mutations
What are the advantages of sequencing over microarrays.
Sequencing has greater coverage, greater versatility (single wells in microarrays can search for a single mutation), it can identify novel variants.
What is an advantage of transcriptome sequencing? (RNA-seq)
Good for certain cancers that are identified and stratified by their transcriptional profile.
What is the use of methylome analysis?
For classification of tumours and biomarker discovery
What is the use of ChIP-seq
To profile chromatin. It marks DNA-protein interactions across the genome
What is the benefit of liquid biopsies in terms of DNA fragmentation?
Circulating nucleic acids tend to be fragmented already so you don’t need to shear it.
Why does fragmented DNA get used for DNA sequencing?
Sequencing technologies only start sequencing from the ends of the molecule. They are unable to sequence therefore over great length before the quality of the base readout begins to degrade. Fragments of DNA prevent this.
In whole exome sequencing, how are exons captured from the rest of the DNA fragments?
Design synthetic RNA oligos which are tagged with biotin. DNA from the library is separated into single strands and then hybridised with the oligos that are exon specific. The biotin tag can then be eluted by Biotin binding beads.
How is RNA sequencing performed?
1) get rid of DNA using DNAses - this leaves you with pure RNA.
2) Fragment RNA
3) Reverse transcriptase forms RNA/DNA duplex which is then transformed into cDNA after the removal of RNA.
4) Add primers via ligation
5) Either perform PCR amplification if required or go straight to sequencing of DNA. This represents the amount of transcription certain genes are undergoing.
What is bisulfite sequencing?
This is looking for methyl-cytosine found on DNA.
1) Conversion of unmethylated cytosine into uracil following incubation of DNA with bisulfite.
2) Fragment ssDNA and undergo random-primed DNA synthesis.
3) Sequence DNA and compare against original genome. Areas where there are is a direct match of cytosine bases will be methyl-cytosine. Where cytosine has been changed to thymine = unmethylated cytosine. This can give a methylation profile of certain genes.
How does SANGER sequencing work?
1) DNA fragmentation
2) In vivo cloning and amplification
3) Denature DNA to produce template strands.
4) Primer addition at 3’ end
5) Add polymerase with dNTPs
6) Add labeled ddNTPs (chain terminating)
7) Capillary electrophoresis of fragments.
8) Order of bases determined by the size of fragment.
What is Illumina sequencing?
1) DNA attached to a flow cell
2) Amplification of DNA involves bridge formation, followed by denaturation, forming clusters.
3) Sequence is built up over multiple cycles involving fluorescent labelling to see which base is in each cluster
