DNA sequencing Flashcards
In DNA
sequencing
we are trying
to find out …
…the order of bases in a strand.
WHat are the uses of dna sequencing?
- To identify genetic mutations, predict disease risk, and prognosis. such as BRCA1 and BRAC2 in breast cancer.
- Infection disease monitoring
Gene cloning - to check your sequence after isolation and/or aplification.
slide 6
What are the two types of DNA sequencing methods?
1) Chain-termination sequencing
2) Next-generation sequencing.
How do DNA polymers grow?
by addition of nucleotides at the 3’ end ie sugar phosphate backbone.
Addition of a nucleotide results in….
…the production of a pyrophosphate molecule (outer two or β and
γ, P-O-P)
What does addition of a nucleotide result in the loss of?
loss of the OH group at the 3’
terminus to form a phosphodiester bond (this
incorporates the α phosphate of the incoming
deoxyribonucleotide).
In growth of DNA polymers, how doe shydrogen bonding occur?
Occurs between nitrogenous bases (adenine, guanine, thymine, cytosine, holding the strands together.
Why does the 3’ end of the growing DNA chain continue growing?
It keeps growing because each nucleotide that’s added has its own free 3’ OH end ( dATP)
If you add a nucleotide in which this OH is
missing the chain will …
… terminate at that point.
Termination of the chain growth is utilised in…
…sequencing - to stop the process of growing the chain.
Nucleotides can be made to achieve termination of chain growth known as…
dideoxynucleotides (ddATP)
DNA fragments can be separated by
electrophoresis according to their ….
….size
In electrophoresis, DNA can be pulled towards a positive fields as it has a…
…-ve charge (phosphate groups), and moves towards the +ve anode during electrophoresis.
in electrophoresis, the larger the fragment, the slower it …
… runs through a gel (DNA ladder).
If you combine DNA chain elongation termination (adding nucleotides with no free OH (ddATP) with the above size separation technique you can…
…order the fragments
and then find out the sequence of nucleotides / bases.
PCR allows….
…strands to build up, adding a base at a time.
If we carry out PCR with
dideoxynucleotides (ddATP)
(no free OH) we can…
… make the PCR terminate.
If we ‘spike’ in small amounts of one of the bases
as the dd format (e.g ddATP Adenine - no free OH)
When ddATP Adenine base pairs the PCR …
… terminates, leaving ‘A’ at the end of the sequence.
By running PCR reactions with each of the 4 ddA, ddT, ddG, ddC (dideoxynucleotides), we will force the PCR to…
…terminate when each of the bases are inserted..
Labelling dideoxynucleotides (ddATP), using for example colour / floursecence means that we can then determine …
…. which bae the strand of the amplified DNA ended with.
Sorting by size then reveals…
… the sequence order.
What is capillary electrophoresis?
To sort by size the fragments in the four sequencing reactions are
electrophoresed in denaturing SDS polyacrylamide gel for size separation
slide 12
The bands detected through capillary electrophoresis are transformed into an …
…electropherogram.
What does each peak relate to in an electropherogram?
each peak relates to the particular fluorescent signal that was detected at a certain time during the capillary electrophoresis run
Slide 14
When was the first complete genome published?
2022
To clone a gene and express the protein it encodes, what needs to be checked?
To clone a gene and express the protein it encodes – vital to check that the sequence is correct otherwise the protein will be wrong
Slide 17
Define NGS.
term used to describe a number of different
modern sequencing technologies
What can NGS be used to do?
entire human genome can be sequenced within
a single day
slide 20
Example of NGS Technology ?
illumina Sequencing
slide 22
What is illumina sequencing also known as?
sequencing by synthesis.
Steps of illumina sequencing?
- Fragmentation of DNA
- Immobilisation of DNA fragments – solid support
- Amplification of immobilised fragments
- Reversible terminator sequencing – Illumina sequencing
