DNA Sequencing by Dideoxy-chain termination Flashcards
Why is Dideoxy sequencing a gold standard sequencing technique?
• It is a Very robust technique – with very low error rate therefore highly reliable and accurate compare to all other methods for deriving a DNA sequence.
Hence it is still a “gold standard” technique
Why is Dideoxy chain termination often referred to as Sanger Sequencing?
This is because it was Method developed to sequence DNA in late 1970s by Fred Sanger who subsequently won the Nobel Prize for his contribution to the scientific understanding of the genome
How does Dideoxy chain termination differ from the 1970s?
• In essence the method used today remains the same as that in 1977, whilst the original technique used radio nucleotides and acrylamide slab gels, today the technology has improved, and the technique has been modified to use fluorescent molecules, capillary electrophoresis and is semi-automated. It is this that has enable the sequencing of the human genome.
How many stages are involved in Dideoxy chain termination , what instructment is it carried out by on both steps?
Today on large scale Automation is possible with Sanger sequencing, however this is in a two stage process where samples are prepared by dideoxy chain termination on a large scale using robotics
• But this requires the separation of labelling the DNA and determining the sequence using separate instruments, the latter part is carried out on instruments such as the 37 30
• In most smaller labs the first stage requires considerable manual hands on preparation prior to sequencing
Name some statistics on the method
- The technique has a read length of up to 900 bases, with 99.95% accuracy; which means an error is made 5 times in 10,000 bases
- A system such as we see here is able to Handle 48 or 96 Samples simultaneously and thus >1000 samples per day
- Technique was used to sequence the Human Genome
- Produced 23 thousand million bases of sequence (23Gbases)
- But took 13 years & $2.7 bn to complete
- However even given this Sanger sequencing is still routinely used in health and biomedicine.
- In particular it is the method of choice to confirm the presence of a mutation in clinical or academic context
Describe the steps involved in dideoxy chain termination
It is a multistage process
• this means we have to Produce a template, often this is done by PCR but could be by any number of means
• Next we Perform a sequencing reaction
There are many similarities to PCR, in that the approach also uses a DNA dependant DNA polymerase to make copies of the complementary strand of a DNA template.
• The next step in the process facilities the determination of the sequence itself
In this step, separation of the labelled molecules occurs sorting them by their size and thus length
This is achieved by capillary electrophoresis giving high resolution separation of molecules that differ in size by a single base
• Since individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout
How is dideoxy chain termination similar to PCR?
Why do we not refer to it as PCR reaction?
Why?
• The Dideoxy sequencing reaction is similar
some protocols also cycle through repeated temperature changes and thus use a thermostable enzyme
They thus repeatedly denature, anneal a primer and perform an elongation step
BUT do not refer to this reaction as PCR there is no exponential amplification and no chain reaction
• why? Sanger sequencing only uses a single forward primer –
That means amplification thus is limited and NOT exponential because the complementary product of the reaction does not act as a template for subsequent rounds as in PCR
What are the indivual parts of the process?
• Strand separation • Annealing primer • Extension (elongation) • Chain Termination And if the reaction is cycled these are then simply repeated a number of times
What happens in strand separation and annealing primer
The annealed DNA and primer is mixed with the reaction components
• The starting material is a clonal population of identical molecules.
What does that mean well it could be a PCR product, a plasmid, or sample of your genomic DNA
• To design the primer we need to know something about that molecule as we require a primer which is complementary to a portion to the DNA that is 5’ to the region we want to sequence.
• We then anneal (or hybridise) the primer to the template forming a partially double stranded structure. And as with PCR, the annealing is driven by the molar excess of the primer in a competition with renaturation of the template
• This partially double stranded structure can be recognised by DNA polymerase and as a consequence forms an initiation complex and starts to elongate the primer from its free 3’ OH group
What happens during extension?
What we have within the reaction are the various different components:
• Our template with a primer annealed to it with a free 3’ OH
• All four deoxynucleotide triphosphates
• And DNA polymerases require Mg ions that act as a cofactor
• And of course we require some sort of buffer to provide the correct pH balance
• The consequence is that polymerase recognises the template initiates elongation and extends the primer by adding a nucleotide to the 3’ OH that is complementary to the template strand hydrolysing the triphosphate forming a phosphodiester bond
Give a more specific describtion of how a dNTP base is added?
Why do we need chain termination?
So how does that work you’ll remember from your first year and the earlier the lecture on DNA hybridisation and complementarity
• If we have our primer, the dinucleotide on the slide represents the primer annealed to the template strand via base pairing with its complement
• its terminal 3’OH group can react with the phosphate of a nucleotide triphosphate presented by the polymerase
• The reaction forms an ester bond, which releases inorganic pyrophosphate and hydrogen ions thus elongating the strand
• As we also saw with PCR the reaction releases of hydrogen ions gradually acidifying the reaction,
• Once the base is added the polymerase then translocates along the molecule to the repeat the process
• The polymerase will continue until it runs out of template, the reaction becomes poisoned by acidification or depletion of nucleotides. Therefore we need chain termination
What does chain termination allow us to do?
Chain termination allows us to randomly halt the elongation, this is simply achieved by the inclusion in the reaction mixture of all 4 dideoxy nucleotide triphosphates
What does doing the chain termination step at low molecular ratios allow?
• By doing this at low molar ratios of the ddideoxy to the deoxynucleotides the polymerase will incorporate a dideoxynucleotide with low frequency and thus terminate elongation in a low proportion of elongating strands
For example if we have ddGTP at a 1 thousandth the concentration of dGTP, when the polymerase incorporates a guanine it will have a probability on stopping of 1 in 1000 an alternative way to think about that is 1 in 1000 elongating strands adding a G will terminate and the remainder will continue beyond that point
Since we have all four dideoxynucleotides this means that the molecules produced by the reaction will vary in length according to when a dideoxynucleotide was incorporated In reality the reaction mixture contains billions of copies of the template and as consequence we are able to terminate elongation at every position in the template millions of times
What does chain termination require? (5)
Requires:
- a template strand that extends a primer forming a partial duplex
- free 3’ OH group on the primer
- All 4 Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- All 4 Dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP)
- Mg2+ ions
So why do dideoxy nucleotides cause termination of elongation?
How do we differentiate between the 4 different dideoxynucleotides?
- The answer to that is in the name, ie a dideoxynucleotide has two hydroxyl groups missing one each at the 2’ and 3’ positions of the ribose ring.
- But as it has a normal 5’ triphosphate it may be incorporated by the polymerase all the same
- And The polymerase is unable to differentiate between these molecules so incorporation is simply down to the molar ratio and chance.
• We already know that extension of the chain is dependent upon having a free 3’ OH group, thus by incorporating a modified nucleotide with a missing OH, we prevent further extension of the strand
• Since we have four different dideoxynucleotide in the reaction
we need some means to differentiate between them. For this we modify each by adding a fluorescent label, thus all the chains terminating with a given dideoxynucleotide will fluoresce at a different wavelength (colour)
