Week 22: (A) Introducing Sanger Sequencing, genome sequencing strategies, and next generation (Illumina) sequencing. Flashcards
What is Sanger sequencing ?
sequencing by synthesis method
What do we need for Sanger sequencing?
sequencing primer, nucleotide to create new strand
template DNA, DNA polymerase, some form of detection method
What is used in the radioactive dye determination sequence method?
each combination of 4 bases, (deoxyNTPS) combined with 1 dideoxynucleotides which would terminate the reaction.
What methods did Sanger use to sequence?
dye termination sequencing (radioactive method)
What is the ddNTP labeled with?
fluorophore, diff colour for each ddNTP
What types of ddNTPs are there?
one for all 4 bases
A, G, T, C
How do we detect where ddNTPs terminate the sequence?
capillary electrophoresis
How are the sequences separated in capillary electrophoresis?
sample buffer (+ve change) runs along a narrow glass tube with gel in it. runs to +ve buffer solution. as it runs to buffer solu, it passes through a laser which allows the fluorescent detector to detect what base it has been terminated at
How can the Sanger method fail?
if we get impurities in the solution by sequencing 2 strands at once with the same primer
(will cute both sequences potentially at the same point and cause overlay)
What needs to happen in order to minimise overlay of sequencing but not have to create many sequences, primers etc.?
1) split the genome into manageable chunks (billions to 40,000-200,000 bases)
2) work out which order they are in
3) get the DNA sequence of each chunk
4) put them together and we have a genome sequence.
How do we split the genome into manageable chunks?
use restriction enzyme. find one that is rare cutting (every 50-200kb) cut up into smaller chunks
How do we clone the chunks of DNA sequence?
clone into a BAC vector to give artificial chromosomes
i.e a plasmid that can replicate DNA sequences of that size faithfully
What is the plasmid called that we replicate and amplify chunks of DNA in?
BAC vector
bacterial artificial chromosome
What does transfect mean?
introduce (genetic material) by infecting a cell with free nucleic acid.
What happens after the DNA chunk is inserted into BAC?
it is colonised on an agar plate,
colonies form (each one a diff chunk of the sequence)
put into wet and iron as a single culture
purified
analyses independently
how do we work out which order the DNA genomic sequences are in?
carry out an enzyme digest
use the enzyme digest fingerprints (overlapping regions)
to work out the order
create a physical map
What is it called when we create a physical map from the enzyme digest?
tiling paths
like a jigsaw
What is the stage of getting the DNA sequence of each chunk called?
shotgun sequence a see blow the whole thing a part
What do we do to the genomic sequence in shotgun sequencing?
fragment the genomic sequence into a suitable size ~1200bp
Why do we fragment the genomic sequence into 1200bp?
as the total length is 1400 bp so we will get some overlap when we shotgun the sequence
Where do we put the fragmented genomic DNA?
into a sequencing vector
What type of plasmid do we insert the fragmented genomic sequence into?
sequencing vector normal plasmid flanking the site were we insert out DNA The 2 sites which correspond to standard primer sites use the exact same primer
what is a contiguous?
the sequence assembles without the overlaps
What is the economic problem of Sanger sequencing?
slow an expensive
What is the difference between Sanger sequencing and illumina sequencing?
sanger: sequence all bases in one molecule at a time
Illumina: sequence all molecules the same time
What is the disadvantage to Illumina?
relatively short reads (19bp now up to 300bp)
expensive for single sequence
How does illumine work?
sequence one base at a time in millions of sequences
What are the steps in illumina sequencing?
spread DNA sequence on a microscope slide
really diluted,
amplify DNA by PCR to create a polony
use a common universal primer
can sequence one base and stop then sequence the next base and stop
How do we dilute the DNA sequence for illumina?
take a scalpel and take a bit the DNA sequence at the appropriate band on an agarose gel, put in an epindorf tube.
needs it spread out to see
What is shotgun sequencing?
The method involves breaking the genome into a collection of small DNA fragments that are sequenced individually.
