Fergus - Lecture 1 - part 2 Flashcards
What are the components of a typical sequencing reaction?
DNA template
Synthetic olgionucleotide primers
dATP, dCTP, dGTP, dTTP
Dideoxynucleotide (deoxynucleotide with no O on 4’ C)
DNA polymerase
Buffer
Detection label
How does a sequencing reaction work?
(5)
Denature double strand
Add mix of DNA nucleotides and dideoxynucleotides e.g. ddATP
Most of the time the DNA will add on a normal nucleotide but sometimes it will add on a ddNTP and wont be able to extend further and will result in a termination
However because there is no OH at the 3’ end they wont be able to extend
We can find the position of each ddNTP (ddATP, ddCTP etc) by finding out the sizes of all the terminated products using gel electrophoresis
KNOW DIAGRAM AND GEL
Give some examples of DNA templates
Denatured double stranded templates
Cloning vectors with built in primer binding sites
PCR products
Learn vector in slides
Learn vector in slides
Why is DNA sizing needed?
If you have a 10,000 bp sequence you could design a primer for the first 1,000 bps and keep repeating this but it would takes months
Instead we randomly shear and subclone DNA
What is shearing and how is it used?
Randomly physically chopping up the sequence into loads and loads of smaller overlapping fragments
We then clone these into vectors
We randomly sequence about 100 of these (on the one day)
There will be some overlap but you will have the whole sequence done in a day
What are the four ways of DNA sizing?
Random shearing and subcloning of DNA
Exonuclease III digestion of DNA
Progressive sequencing of DNA
Next generation sequencing
How does exonuclease III digestion work (in DNA sizing)?
KNOW DIAGRAM FROM LECTURE
Exonucleases A and B degrade DNA at different time points to give different sizes of sequences
There might be small amount of overlapping
Write a note on olgionucleotides (in DNA sizing)
(3)
Act as the DNA polymerase priming site
Complementary to known sequence or to a universal primer site in a vector
Can be labelled with radioactive or fluorescent labels to enable detection of sequencing products
Write a note on dNTPs
(4)
Components of DNA
Required by DNA polymerase for chain extension
Supplied at concentrations that will not limit DNA
Nucleotide analogues can be used to avoid DNA structure problems
Give an example of a nucleotide analogue
7-deaza dGTP
Write a note on ddNTP
(3)
Incorporated into extending DNA strand as normal
Once incorporated chain extension terminates
Can be labelled fluorescently for automated detection
Write a note on DNA polymeras
Adds dNTPS or ddNTPS onto the 3’ hydroxyl of an extending strand
T7 (sequenase)
Taq polymerase
Thermosequenase
Name two radioactive labels
35S
33P
What type of electrophoresis is most common in automated sequencers?
Capillary electrophoresis
How does automated sequencing work?
Detection is done by incorporating fluorescent tags into primers
List three different types of automated sequencers
ABI
Lycor
Megabase
Write a note on ABI
(6)
Most commonly used
Can use up to 4 fluorescent tags
Label is on the ddNTP
All 4 termination reactions can be loaded into the 1 well
Laser scans across gel with detection microscope
Capillary electrophoresis
Write a note on Lycor
(4)
2 fluorecent tags
Label is on the primer
2 sequencing reactions (a forward and reverse) require 4 lanes
Dual laser scans across gel with detection microscope
Write a note on megabase
4 fluorescent tags can be use
All 4 termination reactions can be loaded into 1 capillary
How does big dye labelling work?
(3)
Each ddNTP is labelled with one of the four fluorescent dyes
The sequencer can determine which ddNTP terminated the reaction and therefore can determine which nucleotide base (A, T, G, C) is at that point
4 reactions can be performed in the one tube
What was the sequencing strategy of the original human genome project?
(9)
It was done one chromosome at a time
They chopped the chromosome into chunks
These fragments were inserted into plasmids, bacterial artificial chromosomes (BAC) and cloned in bacteria
The fragments are then mapped so its known what region of the chromosome they came from
Each BAC insert is then sequenced
Each BAC is shotgunned into pieces, process repeated several times to give different sets of fragments
Fragments are cloned into small vectors and then sequenced
Sequences are fed into a computer which looks for overlaps
When enough of the fragments have been sequenced the sequence of the original BAC can be assembled -> this is done for all BACs to give a complete chromosomal sequence
What is shotgunning?
Breaking apart a BAC into random fragments of different sizes
What is whole-genome shotgunning?
This is when a whole genome is sequenced without the use of BACs i.e. the whole chromosome is shotgunned
What are the main challenges for large scale sequencing?
(2)
High resolution physical map required
Highly redundant clone library required
What is next generation sequencing?
The ability to sequence whole genomes in a single run
What automation is used in next generation sequencing?
(3)
Roche 454
Illumina
Life technologies
How does next generation sequencing work, what is it based on?
(3)
Fragment a genome
Separate the fragments on to some sort of solid support e.g. beads or chips
We detect the termination products via fluorescent
What are the three types of new generation sequencing?
Whole genome sequencing
Whole exome sequencing
Targeted sequencing
Write a note on whole genome sequencing
(5)
We will find everything including junk DNA
30x coverage (every base sequenced 30 times)
Lots of overlap
Lots of sequence variations e.g. deletions
Very wasteful in diagnostic labs
Write a note on whole exome sequencing
(4)
Captures fragments related to genes
Can sequence up to 50 or 100 times depth
We detect all polymorphisms in protein coding regions
Cost effective
Write a note on targeted sequencing
(4)
Targets fragments for individual disease
Can sequence up to 500 times depth
e.g. can look at just the exons of cystic fibrosis
Most cost effective -> can do 50 genes in 8 individuals in one run
Write a note on the roche system
(9)
Amplification of fragments in an oil aqueous emulsion
Fragment the genome
Join adapters to end of fragments
Mix fragments with excess micro beads -> only one bead binds to one fragment
Mix beads with PCR reagent (=micro-reactors)
Perform emulsion PCR
Break down emulsion so we are left with only the amplified single fragment
Pipette them into PicoTiter plate -> one bead per well
Load with sequencing reaction
How does the illumina system of whole genome sequencing work?
(2)
A chip is used instead of a bead
This leads to bridge amplification -> amplify the original fragment and copy it multiple times
How is medial genetics different to other medical diagnostics?
You can tell someone that they will die of a disease before they even have symptoms
What is a pedigree analysis?
Whole family genetics
Give some examples of human genome databases
(3)
NCBI -> National Centre for Biotechnology Information
Ensemble -> collab between European Bioinformatics Institute and Wellcome Trust
UCSC Genome browser (small academic group)
