DNA Sequencing Flashcards
What is the dideoxy chain termination method (DNA sequencing)?
- Often called Sanger Sequencing
- Method developed to sequence DNA in 1977
- Developed by Nobel Prize Winner Fred Sanger
- In essence the method used today remains the same.
- Technology has improved, the technique has been modified and semi-automated.
- Very robust – with low error rate therefore highly reliable –> Hence it is still a “gold standard” technique.
It was the method used to sequence the entire Human Genome and produced 23 thousand million bases of sequence (23Gbases). This took 13 years to complete and was a $2.7bn investment in genomics.
Describe how DNA sequencing has become automated.
A machine commonly used is the ABI 3730.
The samples are prepared by dideoxy chain termination on a large scale by robotics. It has a read length of up to 900 base pairs and 99.95% accuracy (An error occurs 5 times in 10000.).
It handles 48 or 96 samples simultaneously, and can go through more than 1000 samples a day.
But, it only performs the separation of labelled DNA and determines the sequence - it requires considerable hands-on manipulation.
Define dideoxy chain termination.
Traditional dideoxy nucleotide sequencing by strand termination is a method that uses an enzyme called DNA-dependant DNA Polymerase to make copies of the complementary strand of a DNA template.
It uses a separation step in which molecules produced are sorted by size, and, since individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout.
What are the steps in dideoxy chain termination?
- Generating a Template:
This can either be a clone (plasmid) or an amplicon (from PCR). - Sequencing Reaction:
DNA Polymerase makes multiple copies of the template. - Separation on size:
This is done using capillary electrophoresis. - Detection of Reaction Particles:
Here, sequential detection of the terminating nucleotide to identify the base occurs. - Readout of the sequence:
This is where the sequence is reconstructed.
The individual molecules produced make up a mixed population terminated by a particular dideoxynucleotide –position terminated is determined by the template sequence.
How is dideoxy chain termination different to PCR?
The dideoxy sequencing reaction is similar to some protocols in PCR, such as that they cycle through repeated temperatures. However, dedioxy sequencing only uses a single forward primer - this means that amplification is limited (linear) and not exponential.
They both use a DNA Polymerase (if cycling is performed, a thermostable polymerase would be necessary).
What are the different steps in the sequencing reaction in dideoxy chain termination?
- Strand separation
- Annealing primer
- Extension
- Chain Termination
How is the primer designed for the first steps of the process?
The primer must be designed 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.
Expand on the strand separation and annealing primer stages of the sequencing reaction.
The first two steps are strand separation and annealing primer.
- The annealed DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides. A labelled single stranded oligonucleotide (primer) is bound to the template.
- The starting material is a clonal population of identical molecules.
This could be a PCR product, a plasmid, or sample of your genomic DNA. - The polymerase recognises the DNA and then forms an initiation complex and commences elongation from the 3’ terminus (free 3’ OH group) of the primer in a 5’ to 3’ direction.
What is involved in the extension stage of the reaction.
The third step of the sequencing reaction is extension.
This occurs by the action of DNA polymerase. It requires:
- a template strand that extends beyond a primer
- free 3’ OH group on the primer
- all 4 deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions (cofactor for DNA Polymerase)
- Buffer to provide correct pH balance.
What happens in the extension stage of the reaction?
The 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 and forming a phosphodiester bond (ester bond). Inorganic pyrophosphate and H+ ions are released, thus elongating the strand.
Similar to PCR, the H+ ion release gradually acidifies the reaction.
Once the base is added, the polymerase then translocates along the molecule to the repeat the process.
What would happen if there wasn’t a chain terminating step?
Without this part the polymerase would continue adding bases until the enzyme ran out of template, the reaction became poisoned by acidification or depletion of nucleotides halted elongation. In which case we’d have no means of determining the sequence.
Expand on the chain termination step of the sequencing reaction.
The 4th step of the sequencing reaction is chain termination.
DNA elongation is terminated by the addition of a dideoxynucleotide (ddATP, ddGTP, ddCTP, ddTTP). Dideoxynucleotides prevent elongation.
As the enzyme encounters a particular nucleotide (e.g. guanine) in the sequence, it picks out a complementary cytosine and incorporates it into the elongating strand. However, the reaction contains both dCTP and ddCTP; if a dideoxy molecule is incorporated into the strand, elongation is terminated, but if a deoxy molecule is incorporated, elongation continues.
Since we have all 4 dideoxynucleotides, the molecules produced by the reaction will vary in length according to when the dideoxynucleotide is 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.
Why do dideoxy nucleotides cause termination of elongation?
Dideoxynucleotide has 2 hydroxyl groups missing, one 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. The polymerase is unable to differentiate between these molecules so incorporation is simply down to the molar ratio and chance.
The 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).
Explain the implications of adding the dideoxy molecules into the sequencing reaction.
Products where a ddCTP (eg) is incorporated represent all positions within the sequence where a ‘cytosine’ occurs.
Since all four labelled dideoxy nucleotides are present in the reaction, the population of molecules produces represent all possible reactions in the sequence from the same point to the end.
Reaction products vary in length and are terminated by ddNTP. Ordering these molecules by size allows us to determine the sequence of the new strand.
Describe the size separation by gel electrophoresis.
The nucleic acid passes through a gel matrix by applying a voltage across two electrodes.
Negatively charged nucleic acid will migrate towards the positive electrode.
The matrix retards the molecules according to their size. Those that are larger are retarded to a greater extent and as a consequence move through the matrix more slowly.
As the population of molecules are separated at single base resolution If we place a sensor at the terminal end of the capillary and monitor the fluorescence as the population of labelled molecules pass this point we may reconstitute the sequence.
