DNA sequencing Flashcards
What did Maxam and Gilbert develop?
Sequencing.
When did Maxam and Gilbert develop sequencing?
In 1977-80.
How was ‘sequencing’ characterised?
The first widely-adopted method for DNA sequencing.
To what was ‘sequencing’ based?
On nucleobase-specific partial chemical modification of DNA.
On subsequent cleavage of DNA backbone at sites adjacent to modified nucleotides.
What was DNA Kinase needed about?
To add radioactive 32P to the 5’ end of the strand.
Why was DNA Kinase needed to add radioactive 32P to the 5’ end of the strand?
To sequence the DNA strand.
How many different reactions wee run on a polyacrylamide gel?
4.
Where di DNA sequence read up?
Across the 4 lanes of the 4 different reactions.
By what was the Maxam-Gilbert sequencing replaced?
By the Sanger method.
What was the function of the Maxam-Gilbert method of DNA sequencing?
Destroying each of the 4 bases in separate reaction.
Running the 4 separate reaction on acrylamide gels.
Why was the Maxam & Gilbert method of DNA sequencing running the 4 separate reaction on acrylamide gels?
To read off the sequence.
By who was the ‘Sanger sequencing’ developed?
Sanger et al.
When was the ‘Sanger sequencing’ developed?
In 1977.
What did the ‘Sanger sequencing’ become?
The most widely-adopted method for DNA sequencing.
Until when was the ‘Sanger sequencing’ the most widely-adopted method for DNA sequencing?
Until 2010.
On what was ‘Sanger sequencing’ based?
The incorporation of chain-terminating dideoxy nucleotides (ddNTPs) by DNA polymerase.
What do the ddNTPs lack?
The 3’-OH.
What happens when the ddNTPs have been inserted by DNA polymerase?
No further elongation occurs.
By what can fluorescent ddNTPs be detected?
A laser.
How was the fluorescent ddNTPs characterised originally?
Very expensive method.
How is the fluorescent ddNTPs method characterised now?
Very cheap.
Efficient procedure.
What does the sequencing mixture contain?
Normal dNTPs + a lower concentration of fluorescently-labelled ddNTPs.
What is DNA Polymerase continually adding?
dNTPs.
Until when is DNA Polymerase continually adding dNTPs?
Until it adds a fluorescent ddNTP.
What does a fluorescent ddNTP stop?
Further DNA synthesis.
What happens to the fragments after the sequencing reaction?
They are electrophoresed.
Where are the fragments electrophoresed, after the sequencing reaction?
In a capillary tube.
By what are the fragments read off in a capillary tube, after the sequencing reaction, and how?
By a laser.
One-by-one.
What did the early method of sequencing needed?
32P-labelled primers/32P-labelled ddNTPs.
Where were the reactions performed, in the early sequencing method?
In separate tubes.
Where were the reactions of sequencing run after they were performed in separate tubes, in the early method of sequencing?
On gels.
What is an example of gels where sequencing reactions run on after they are performed in separate tubes?
Those used in Maxam & Gilbert sequencing.
From where are DNA sequences determined?
Electrophoretograms.
How are the fluorescent signals shown in electrophoretograms?
With using different colours for each base.
How many peaks should occur at any time in electrophoretograms?
Only one.
Of what are the double peaks the result?
Of a mixed DNA template.
What can the mixed DNA template be?
Diploid.
Contaminated.
For what is sequencing diploid genomic DNA used?
To show if individuals are homozygous/heterozygous at a particular loci.
What are the major benefits of the Sanger sequencing?
Cost-effectiveness.
Efficiency.
Reliability.
What reads of sequencing are possible?
Long reads up to 800 n.
With what can modifications deal?
With highly-repetitive sequences.
What was an ideal method to show the sequence of relatively small fragments of DNA?
Over-lapping reads from different primers and different DNA fragments .
Why were the over-lapping reads from different primers, an ideal method to show the sequence of relatively small fragments of DNA?
Because they rapidly build up the sequence of a larger region.
What is a PCR-based Sanger sequencing?
A very fast way to confirm the sequence of a small section of DNA.
What does a PCR-based Sanger sequencing do?
Confirms cloning.
Diagnostics.
What does the ‘Next Generation (NGS) sequencing’ include?
New techniques.
New technologies.
Where are the new techniques and technologies based on?
High-throughput approaches with massive parallel sequencing.
Why is the ‘Next Generation sequencing’ based on high-throughput approaches with massive parallel sequencing?
To generate final sequences of more than 1Mb at a time.
How are may techniques aim to show the sequences of lots of small fragments?
All at once.
In what can the sequences of small fragments combined?
In silico.
Why can the sequences of small fragments be combined in silico?
To generate the sequence of very large fragments.
What is the advantage of NGS?
In one processing period huge amounts of DNA sequence can be obtained.
When is the advantage of NGS only cost-efficient?
If near-genome sized sequencing is required.
What does high-quality DNA produces when it is prepared and fragmented?
Smaller pieces.
What does high-quality DNA produce when it is amplified?
Greater copies of target DNA.
What does an amplification of DNA include?
The use of ‘linkers’/’adaptors’.
How are the greater copied of target DNA sequenced?
By using the by-products of DNA synthesis (PPi).
Why are the greater copies of target DNA sequenced by using the by-products of DNA synthesis?
To produce light (pyrosequencing).
Addition of fluorescent primer (sequencing-by-ligation).
Change in fluorescent colour.
Production of H+ (sequencing-by-synthesis).
When where the number of different versions of pyrosequencing developed?
In 1993.
1998.2005.
On what does pyrosequencing rely?
On light detection.
On what is light detection based?
On a chain reaction.
How is the light detected?
By using the pyrophosphate released during DNA synthesis by DNA Polymerase.
How is the light produced?
By using Sulfurylase and Luciferase.
By what are solid array, wells or beads flooded?
By each dNTP.
Why was the flash of light recorded?
To show the DNA sequence.
Of what was the bead-based system capable?
Of sequencing 400-600 megabases of DNA per 10-hour run.
Who developed the ‘Illumina Sequence-by-synthesis sequencing’ first?
Solexa.
By who was ‘Illumina Sequence-by-synthesis sequencing’ acquired?
Illumina.
On what was ‘Illumina Sequence-by-synthesis sequencing’ based?
On reversible dye-terminators.
In what was ‘Illumina Sequence-by-synthesis sequencing’ developed?
In a wide range of sequencing-based technologies.
Which were some of the sequencing-based-technologies?
Transcriptomics.
Methylation profiling.
DNA-protein interactions.
What happens to DNA after purification?
It is tagged?
How is the process of DNA tagging named?
Tag mentation.
How was DNA tagged?
By using transposase.
What does transposase cut?
It cuts randomly the DNA into short fragments.
What is added to either end of cut DNA fragments?
Adaptors.
Where are the adaptors attached?
To a solid surface.
What happens to the adaptors attached to DNA fragments after they attach to a solid surface?
They are amplified.
Why are adaptors amplified?
To provide more template DNA.
What does DNA Pol add?
Fluorescently-labelled dNTPs.
How does DNA Pol add fluorescently-labelled dNTPs?
With a reversible terminator.
How are fluorescently-labelled dNTPs detected?
By a laser.
What do cycles of removing the terminator and adding new dNTPs provide?
Sequence information.
What happens in ‘Illumina Sequence-by-synthesis sequencing’?
Hundreds of thousands of individually-labelled templates are ‘read’ by laser and are linked to a solid support.
What is ‘Ion semiconductor sequencing’?
A method of sequencing.
On what is the ‘Ion semiconductor sequencing’ based?
On the detection of hydrogen ions released during polymerization of DNA.
By what are the hydrogen ions detected during polymerization of DNA developed and when?
By Ion Torrent Systems Inc.
In 2010.
How is the sequencing based on the detection of hydrogen ions released during DNA polymerization named?
‘Sequencing by synthesis’.
By what is the release of hydrogen ions detected?
By an ion-sensitive field-effect transistor (ISFET) ion sensor.
What are the major benefits of ion semiconductor sequencing?
Rapid sequencing speed.
Low upfront and operating costs.
What are the major limitations of ion semiconductor sequencing?
Dealing with long repeats of the same base.
Relatively short read lengths.
How short are the read lengths of ion semiconductor sequencing?
Approximately 400 n per read.
What are the ‘ion sequencing steps’?
Microwells on a semiconductor chip.
What do the micro wells of ion sequencing steps contain?
Many copies of one single-stranded template DNA molecules.
What happens to the DNA Pol in ion sequencing steps?
They are sequentially flooded.
How are the DNA Pol sequentially flooded?
With unmodified dNTPs.
What happens if an introduced dNTP is incorporated into the growing DNA strand by DNA Pol?
A H+ ion is released.
What does the H+ ion released in the reaction release?
The pH of the solution.
By what is the change of the pH of the solution detected?
By an ISFET sensor under each well.
What happens to the unattached dNTPs before the next cycle, when dNTP is introduced?
They are washed.
What will the DNA Polymerase produce every time a dNTP is successfully added to growing chain of DNA?
A H+ ion + pyrophosphate.
What do modern high throughput sequencing projects use?
NGS technologies.
Why do the modern high throughput sequencing projects use NGS technologies?
To rapidly produce a draft meeting.
To add reliability to all base pair positions.
What does the Ion Torrent produce?
Fast drafts with low quality.
What does the Illumina produce?
Better-quality sequences with short reads.
By what is further confirmation of sequences done?
By PCR-based Sanger sequencing.
What happened to high throughput sequencing costs?
They have dramatically fallen.
What is it a fact about the use of high throughput sequencing?
Very cheap to use through specialist sequencing centres.
Where can high throughput sequencing be more efficient than PC-based Sanger sequencing?
When there are multiple small samples to analyse NGS.
What are the characteristics of High throughput sequencing?
Efficient.
Fast.
What is it easier to do due to the fact that high throughput sequencing is efficient and fast?
To ‘sequence everything’ and then use bioinformatics to see what is in the sequences.
For what is sequencing and then bioinformatics easier?
For viruses.
Pathogens.
