Sequencing Flashcards
How does sanger sequencing work (1st gen seq tech)
DNA sequencing developed by Frederick sanger and colleagues in 1977 involving chain termination method to determine nucleotide sequence in DNA fragment.
Steps-
DNA is amplified using PCR
DNA is fragmented using RE
Sequencing reaction is set up in 4 tubes each containing 4 deoxynucleotides (dATP, dGTP, dTTP, dCTP), DNA polymerase and a small amount of modified nucleotide (ddNTP, where N is a base and lacks a 3’ hydroxyl group)
As DNA polymerase incorporates the ddNTP the lack of the 3’ OH group leads to termination as no more nucleotides can be added.
The fragments are then separated by size using gel electrophoresis and are detected using a fluorescent or radioactive label on the ddNTP
How does Maxam-Gilbert sequencing work
Obtain single stranded DNA
Label at 5’ end with radioactive Phosphorus 32
Cleave at specific nucleotides to get different sized fragments
Use electrophoresis through high resolution acrylamide gels
Deduce DNA sequence using labeled DNA
How does next gen sequencing work (2nd gen seq tech)
Genomic DNA is sheared and adaptors are ligated on
The fragments are hybridised onto a solid surface and amplified to form clusters (denaturation and forming of ds repeatedly)
Incorporate labelled nucleotides and highly sensitive cameras record the sequence of lights
Improvements-
Lots of clusters means millions of bases so errors become more insignificant so more accurate and very efficient.
How does 3rd gen seq technologies work
PacBio seq-
Direct reading of single molecules (very fast and cheap but high error rate)
Well containing 1 DNA molecules and a polymerase and when a base is added SMART cells read the illumination and emission coming off (very sensitive)
Oxford nanopore-
Synthetic protein pore where single DNA molecule passes through and electrical changes are detected
Very small so portable (taken out in the field)
How has sequencing helped metagenomics
Metagenomics-
Sequencing DNA of whole environments of whole populations
Reduced cost of NGS have allowed comprehensive studies on microbial communities
How has sequencing used for single cells
Microfluidics can be used to isolate a single cell from an environmental sample or laser capture microdissection used for organ tissue.
The single cell then is isolated and DNA is extracted and amplified using whole genome amplification.
Then these are used to create libraries and then further sequencing through NGS.
Bioinformatic analysis are done to read mapping, variant calling, gene expression quantification.
Describe how hybrid assembly can be used to resolve sequencing ambiguities
Combines short accurate reads from 2nd gen seq technologies with long leads accurate reads from 3rd gen seq to leverage the strengths from both
Short reads correct errors in the long reads and resolve repetitive regions.
Long reads provide valuable info for scaffolding and resolving complex genomic regions.
Tools like unicycler can do this
What are the techniques used to characterise promoters
Reporter gene assays-
Determines where promoters are by ligating DNA containing promoter sequence to reporter gene (GUS)
You can then compare GUS activity with mutants of the promoter to see how much of it is actually important
Chip assays-
Chromatin immunoprecipitation can be used to identify the binding of RNA polymerase 11 and transcription factors to promoter regions.
Provides insights into regulation of transcriptional initiation.
What are the techniques used to characterise the start/end points of transcription
RNA seq-
Can be used to map the 3’ end of transcripts to identify termination sites and transcript isoforms
Transcriptional start sites mapping-
TSS mapping techniques like cap analysis of gene expression (CAGE) and 5’ rapid amplification of cDNA ends (5’ RACE) are used to precisely map the start points of transcription.
How DNase protection assays are used to determine DNA-protein binding sites
DNA fragment is incubated with protein of interest. DNase 1 is added.
The protein binds to specific DNA sequences and protects them from DNase 1 digestion so is separated from the free DNA. The protected DNA fragments are then analysed by gel electrophoresis where the missing bands represents the binding sites.
How gel-shift assays can be used to determine DNA protein binding sites
DNA fragments is incubated with protein and then DNA-protein complex is separated from free DNA using gel electrophoresis.
The DNA-protein complex migrates more slowly than free DNA due to increase mass and altered charge so visually you get a shift in position of the DNA band on gel
