DNA Sequence Flashcards
What is dideoxy chain termination?
Often referred to as sanger sequencing. It is a method developed to sequence DNA. As technology has advanced the technique has been improved and modified. It is a very robust technique with a low error rate thus highly reliable – gold standard.
How does sequencing by dideoxy chain termination work? (brief summary)
It is a multistage process.
- First, a template is produced. This is often done by PCR.
- Next, perform an enzymatic sequencing reaction. Similar to PCR as it uses a DNA dependent polymerase to make copies of the complementary strand of a DNA template.
- Next step is size the labelled molecules. products are separated according to size by capillary electrophoresis (giving high resolution separation of molecules that differ in size by a single base).
- Then, there is a sequential detection of reaction products. (detection of the terminating nucleotide to identify the base).
- Finally, the sequence is readout. Since individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, the original sequence can thus be reconstructed from the readout.
What is the similarities/differences between dideoxy sequencing and PCR?
Some protocols also cycle through repeated temperature changes and thus use a thermostable enzyme (hence uses DNA polymerase). They thus repeatedly denature, anneal a primer and perform an elongation step.
However, there is no exponential amplification and no chain reaction (unlike PCR). This is because Sanger sequencing only uses a single forward primer – thus means amplification 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 4 parts of sanger sequencing?
Strand separation
Annealing primer
Extension
Chain termination
What happens during strand separation and annealing the primer?
The annealed DNA and primer is mixed with the reaction components including both dideoxy and deoxy-nucleotides.
The starting material is a clonal population of identical molecules.
A single stranded oligonucleotide (primer) is bound/annealed to the DNA 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.
DNA polymerase then recognises the partially double stranded DNA structure and as a consequence forms an initiation complex and starts to elongate the primer from its free 3’ OH group.
How does the Extension part work?
The primer is 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. Similar to PCR, the reaction releases of hydrogen ions gradually acidifying the reaction. Once the base (dNTP) is added the polymerase then translocates along the molecule to the repeat the process.
What is required for Chain termination?
-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.
Chain termination allows to randomly halt the elongation. It is achieved by the inclusion in the reaction mixture of all 4 dideoxy nucleotide triphosphates. By doing this at low molar ratios of the dideoxy to the deoxynucleotides the polymerase will incorporate a dideoxynucleotide with low frequency and thus terminate elongation in a low proportion of elongating strands.
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.
How is DNA elongation terminated?
DNA elongation is terminated by the addition of dideoxynucleotide. It 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.
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, they are differentiated between by adding a fluorescent label, thus all the chains terminating with a given dideoxynucleotide will fluoresce at a different wavelength.
Summarise elongation and chain termination.
Upon mixing the reaction the polymerase will commence elongation from the 3’ end of the primer adding complementary nucleotides.
As the enzyme encounters a particular nucleotide eg a guanine in the sequence it acquires a complementary cytosine and incorporates it into the elongating strand.
However since the reaction mix contains both dideoxy and deoxycytosine, which the polymerase cannot discriminate between.
If a dideoxy molecule is incorporated into the strand elongation is terminated however where a deoxycytosine is incorporated elongation continues.
How is size separation carried out by gel electrophoresis?
The nucleic acid passes through a gel matrix by applying a voltage across two electrodes.
Negatively charged nucleic acid migrates 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.
The sequence is thus determined by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.
What is DNA sequencing by dideoxy chain termination used for?
- To confirm all types of mutation – silent, missense, nonsense, truncating, indel and mis-splicing.
- To identify HIV haplotypes resistant to anti-retrovirals to determine therapy HAART
- In research for gene sequencing.
- Used in clone or PCR Amplicon sequencing to confirm a clones sequence or site-directed mutagenesis.
- Confirmation of causative variants associated with genetic disease following GWAS.
