DNA sequencing Flashcards
Dideoxy chain termination developed
In 1977 by Fred Sanger, also called Sanger sequencing - the gold standard for low error rate
Accuracy is
99.95%
Process of sanger sequencing
- Template strand separation and replicated by DNA polymerase before annealing labelled oligonucleotide primer, forming initiation complex.
- Mixed population is terminated by particular dideoxynucleotide, leading to varying product lengths. Sequential detection of terminating nucleotide at particular position determined by template sequence. Missing OH on 3’ prime end prevents further elongation.
- Size separation of products by capillary gel electrophoresis.
- Reconstruction of original sequence in readout of sequence by ordering molecules by size. All positions within the sequence are incorporated with a dideoxy/deoxynucleotide.
DNA dependant DNA polymerase
- Requires template strand that extends beyond primer.
- Free OH group on primer.
- All deoxynucleotide trisphosphates. (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions.
Last step dideoxy chain termination
All 4 Dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP)
• Still routinely used for confirmatory tests for specific genetic mutations (excepting low frequency mosaicism).
• Identifying HIV haplotypes resistant to anti-retrovirals (HAART).
• Used in research (gene sequencing, cloning, candidate gene studies, confirmation studies).
• Confirmation of causative variants associated with genetic disease following association study
• “Walking” a gene to identify a causative mutation in candidate gene studies
• Clone or PCR Amplicon sequencing to confirm a clones sequence or site-directed mutagenesis
• Mammalian and Pathogen Gene sequencing
Sequencing using DNA polymerase
• DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
- the polymerase commences elongation from the 3’ terminus
- as the enzyme encounters a particular nucleotide in the sequence it picks out and incorporates a complementary nucleotide into the elongating strand.
- If a dideoxy nucleotide is incorporated into the strand, then elongation is terminated otherwise elongation continues
Varying lengths of products
- Products where a ddCTP is incorporated therefore 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 produced represent all possible positions in the sequence from the same point to the end.
- Ordering these molecules by size allows us to sort the molecules accorded by the order the terminal nucleotides were incorporated into the elongating strand and thus reconstitute the sequence of the new strand. this is illustrated by the animation
- Reading the fluorescence of the molecule allows us then to determine the sequence and reading the sequence from the 5’ end in this instance we have ATG TAA CGG CTA T……………..
Size separation can be done 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
Determining the sequence
The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.
Sequencing using DNA dependant DNA polymerase
• Measurement of fluorescence generates a trace and base calling is automated
Pcr and dideoxy chain termination
- Similar but some protocols cycle through repeated temperatures BUT only uses a single forward primer - 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
- Dideoxy chain termination also uses a DNA polymerase If cycling is performed a thermostable polymerase so would be necessary and is usually used
