Lecture 3: Studying Human DNA: DNA sequencing (not finished) Flashcards
what san example of a sequence polymorphisms ?
single nucleotide polymorphisms (SNPs)
what does DNA sequencing determine?
DNA sequencing determine the precise order of nucleotides.
is there more than one method to sequence DNA?
yes
whats included in First generation sequencing?
- Chain-termination method - Chemical cleavage method
what is second generation sequencing now also known as?
massively parallel sequencing (MPS)
whats included in second generation sequencing?
Pyrosequencing
Ion torrent
Reversible terminator sequencing
SOLiD sequencing by ligation
Third and fourth generation DNA sequencing uses what?
unamplified DNA.
who and when was Chain-termination method discovered?
Fred Sanger in the 1970s – Sanger sequencing.
what does polyacrylamide gel electrophoresis do?
fragments that differ by a single nucleotide length can be separated.
what are the components of Chain-termination method?
- DNA molecule to be sequenced (target)
- Oligonucleotide primer to target the internal starting point
- DNA polymerase for extension
- 4 Deoxyribonuceloside triphosphates (dNTPs – dATP, dTTP, dCTP and dGTP.)
A small amount of what is added during the Chain-termination method?
dideoxynucleoside triphosphates are added (ddNTPs)
whats the difference between ddNTP compared to a dNTP?
sugar is dideoxyribose in ddNTP rather than deoxyribose.
With dideoxyribose the 3ʹ carbon lacks what?
the hydroxyl group.
How does a ddNTP block further elongation?
lack of a free hydroxyl group a connection cannot be formed with the next nucleotide
what are the first three steps of Chain-termination method?
• DNA is denatured to produce single stranded DNA.
• Oligonucleotide primers anneal and with DNA polymerase chain extension
occurs with dNTP building blocks.
• ddNTPs are also added that can be labelled according to the type of base.
what are the 4th, 5th and 6th steps of Chain-termination method?
- chain termination occurs.
- These products can then be separated by size using electrophoresis.
- For CE The fragments are detected using a fluorescent detector that can discriminate each labelled ddNTP.
in Chain-termination method, what does the strands 3’ end have?
common 5ʹ end but the 3ʹ end will be variable and differ by a single nucleotide length.
In polyacrylamide gel electrophoresis where will you find shorter fragments?
near top of the gel
only ___ddNTP per tube.
one
Capillary Electrophoresis can be done in?
a single tube
In Capillary Electrophoresis what happens to dNTPs?
they are labelled with a fluorescent marker.
in Capillary Electrophoresis what happens with a laser?
A laser excites the fluorophores and the fluorescent
signal is recorded. can be read.
in Chain-termination method how many strands are copied ?
Only one strand is copied unlike PCR.
where is the Purified DNA template for sequencing found?
obtained from molecular cloning or PCR.
If using a PCR product for sequencing what primers can be used?
the same PCR primers
can be used or an internal primer
why do genomes need to be sequenced in sections and multiple times ?
because o the limit this needs to be done to identify errors.
what needs to happened to the shorter sequenced fragments for the larger genome sequence to be determined?
need to be assembled in the correct order
what is the shotgun method?
sequenced fragments can be examined for overlapping sequencing to assemble
what genomes have been assembled by this de novo sequencing shot-gun approach ?
prokaryotic smaller genomes
what is Resequencing ?
A less computer intensive approach is Resequencing where a reference genome from the same species or similar species (de novo) is used.
why are multiple reads done?
to deduce sequence and errors
To ensure that errors are identified at least at least two many fold sequence depth or coverage is
required?
5
what can help deduce sequence errors?
Using a reference genome can help with assembly, making it quicker and more
accurate.
when was the first draft of the human genome project completed?
2001
when was the first finished sequence ?
2003
why is each region sequenced multiple times?
to enable an accurate sequence
identifies error in individual sequence reads.
studying human genomes would be _____ and ______ if using this chain termination method.
slow
costly
how long was chain termination the standard sequencing method?
30 years
what was the main limitation of chain sequencing method?
sequencing throughput of the this method
when was next generation sequencing introduced?
2005 onwards
what is NGS also known as ?
massively-parallel DNA sequencing (MPS)
what does NG enable us to do ?
enabled thousands or millions of DNA fragments to be sequenced in parallel in a single experiment reducing the cost.
Whats good about NGS?
individual reads are much shorter but the overall throughput is much greater.
whats the main difference between chain termination and massively parallel DNA sequencing?
MPS can sequence millions of DNA fragments simultaneously.
MPS platforms fit into two broad categories which are?
- Sequencing of PCR products, sequencing fragments of 35 – 800 nucleotides (nt) with a sequencing throughput of 1000 to a million Mb of DNA.
- Sequencing of unamplified single-molecule sequencing, sequencing fragments thousands of nucleotides long but with a lower sequencing throughput of 100-1000 Mb.
Next gen sequencing workflow?
- extract DNA from patient sample
- sharing of DNA with a method of choice + end rapid fragments
- ligation of specific adaptors+ ligation of barcodes for multiplex beads
- library selection and purification using magnetic streptavidin beads
- amplify using PCR
After the DNA is broken down by sonication to small fragments lengths then what happens?
it is then immobilised to create a DNA library
How is a DNA library created for NGS ?
- using oligonucleotides and adaptors bound to a solid glass
support - Or the use of metallic beads coated with the protein streptavidin (SA) with a biotin labelled adaptor.
What is the final step for preparation of sequence library?
PCR amplification of the immobilised DNA fragments to produce a sufficient number of copies to be sequenced.
IMMOBILISATION OF DNA FRAGMENTS GLASS
DO IT
