Lecture 11 - DNA Sequencing Methods Flashcards
How do you sequence from synthesis?
from a primer incorporating radioactive label and base-specific termination by dideoxyinosine
What does a sanger sequence sequence?
molecularly cloned DNA’s
How can you sequence using chemical degradation?
Base-specific chemical cleavage of radioactively 5’ end-labelled DNA
What is the maxam-Gilbert method used for?
specialised purposes, such analysing DNA-protein interaction
What do both the Sanger and the maxam-Gilbert method rely on?
fractionation of ssDNA in denaturing polyacrylamide gels to give one nucleotide size difference resolution
In the Sanger method what dNTP is radioactively labelled?
a-P32-dCTP or a-S35-dCTP
What does the ratio of dNTP and ddNTP tell us?
probability of chain termination at each position
Are the principles of dye termination in Sanger sequences the same as he normal Sanger sequence?
Yes
Dye terminator Sanger sequencing - how is this different from the Sanger method? What activates the dye?
ideoxynucleotides each have a different fluorescent label and all 4 reactions run in the same lane of the gel
Laser activates dyes at bottom of gel and fluorescenceis detected and recorded
What do you sequence with in a dye terminator Sanger sequence?
T7
Genome sequencing by the Sanger method - how does this start? Cloning
Genome molecularly cloned as large fragments in a BAC library and overlapping BAC clones identified
Genome sequence by Sanger method - what happens to the BAC?
cut into many smaller fragments and ‘shotgun’ cloned into plasmid vectors
Genome sequencing of Sanger method - how is the plasmid sequence assembled?
Cloned DNA in plasmids sequenced using a universal primer and final sequence assembled from sequence overlaps
Next generation sequencing technologies - what are the common principles?
All rely on methods that sequence DNA directly – no starting molecular cloning or PCR production of individual DNAs
Genomic DNA is reduced to small random DNA fragments representing all the sequences of the starting DNA (DNA fragment library) and these are simultaneously sequenced in a single procedure
How does the average NGS technology work?
DNA reduced to small random fragments by sonification.
DNA fragment library is then modified by adapter ligations and PCR
DNA fragments then immobilise to a solid support so that the locations of individual DNA molecules are separated
What do NGS tech need and where does this occur?
Parallel sequencing in flow cells, occurs at a discrete solid phase clonal amplification and the number of DNA in a colony or cluster sufficient enough to be detectable
What do parallel sequences need to be?
simultaneously monitored by a detector system
What do NGS tech not have to do?
sequencing reactions do not involve separation of products by electrophoresis to generate nucleotide sequence reads
What is the ILLUMINA NGS method:
the principle of sequencing by synthesis using DNA polymerase and fluorescent terminators
How does the illumina NGS work?
sample preparation; cluster generation to amplify individual DNA molecules in situ; sequencing by synthesis with simultaneous imaging to record fluorescence emission; data analysis
What is step 1 of an illumina NGS - English oligonucleotide?
Short single strand oligonucleotides of two defined sequence (P5 andP7) are chemically bonded to the glass slides (tiles) or nanowells in a flow cell to form a dense lawn
What way are ogkionucleotides bonded in illumina NGS?
Short single strand oligonucleotides are chemically bonded via their 5’ends with 3’ hydroxyl ends free and pointing up from the surface
Illumina NGS method - how to adapters bind to the oglionucleotides and what does this do?
Adapters with the same sequence as the bonded oligonucleotides areligated to each end of the sonicated DNA from the genome of interest
Illumina NGS method - how are adapters ligated?
The double-stranded adapters are ligated on in a way that results in allsonicated DNAs having a different adapter sequence on each end
Illumina NGS method - what happens after ligation?
DNA population is denatured and added to the glass slide, the DNA attaches via complementary base pairing to the bonded oligonucleotide that will act as primers for DNA polymerisation
Illumina NGS method - what happens to the density of the attached dna?
Is adjusted so that single DNA molecules are on average attached at well separated positions from one another
Illumina NGS method - after the density of dna has been set what happens to the dna and dNTPs?
flushed through the flow cell and eachsingle DNA molecule is copied in situ by DNA polymerisation initiated atthe 3’ end of the chemically bonded oligonucleotide primers P5 and P7
Illumina NGS Method - what happens to the original DNA
DNA molecule is washed away after denaturation to leave achemically bonded single copy of itsel
Illumina NGS method - what happens to the copy of the DNA after it has been denatured?
renaturation conditions anneals to an adjacent chemically bonded oligonucleotide P5 primer to initiate another round ofDNA synthesis by DNA polymerase - called ‘bridge amplification
Are cycles in beige amplifications repeated many times?
Yes
In illumina NGS when sequencing what happens to one of the strands?
It needs to be removed
Illumina NGS methods - how is the strand removed?
P5 oligonucleotide is chemically cleaved and the black strand removedby denaturation
What does each cluster now contain?
homogeneous population of strands forsequencing using a primer
Are clinal clusters at high density?
Yes
What does synthesis of sequences in Illumina use?
dNTPs with a different fluorescent group for dATP, dCTP, dGTP, dTTP, and areversible 3’ chain terminator block
What can be annoyed to adapter sequences?
Universal primer
What happens after universal primer?
DNA polymerase is added and sequencing is started on all the singlestrand templates in the cluster using the reversible dNTP terminators
What does imagining record?
records the laser activated fluorescence colour at each clusterposition simultaneously – billions of positions
A single fluorescence output is obtained for each cluster and recorded
What happens after imaging?
dye is chemically cleaved away and the 3’end is chemically unblocked by washing the flow cell with chemicalreagents
What is the output of the sequences number of reads referred to?
The depth
What does partly overlapping sequences result from?
from the multiple templates being sequenced across the flow cell
What are the applications of this technology?
Whole genome resequencing, gene expression, chIP sequencing, microRNA delivery, target resequencing.
How do you determine the structure of chromatin?
DNase I hypersensitivity mapping or Chromatin immunoprecipitation
DNase I hypersensitivity mapping - why is this used?
To map open regions of chromatin structure which gives you an idea about promotes ect.
How is DNase I hypersensitivity mapping done?
Restriction enzyme digestion and southern blotting
Why is chIP used?
detecting sequences associated with specific chromatin modifications (e.g.histonemodifications) orbinding of specific transcription factors
How is chIP done?
ImmunprecipitatedDNA is detected byregion-specific PCRprimers
What can NGS do for chromatin?
genome-wide chromatin sampling can be done by NGS sequencing
How can you understand the content of DNA extraction better?
the content of the DNA extracted (‘millions’ of molecules)a short read or pair of reads of sequence is generated from each originalmolecule obtained from the chromatin sampling
How do informatics methods give peaks and analyse the distribution?
Informatics methods ‘count’ the number of reads at each location to give peaks and analyse the distribution of peaks throughout the genome
What can DNase I hypersensitive sites help with?
DNA footprinting
Third generation sequencing - what does the nanopore minION do?
Nanopore sequencing of single molecules
