10. DNA Sequencing Flashcards
What is dideoxy chain termination?
- Often called Sanger Sequencing - This is a technique to seqeunce DNA.
- 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 the molecles 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.
Requirement for amplification of a DNA sequence for sanger sequencing
- Similar to PCR but - some protocols cycle through repeated temperatures BUT only uses a single forward primer - amplification is limited and NOT exponential
- Uses DNA polymerase - If cycling is performed a thermostable polymerase so would be necessary and is usually used
How does dideoxy chain termination work?
1) DNA polymerase makes mutliple copies of the DNA
2) Sorting by size (capillary electrophoresis)
3) Sequential detection of the terminating nucleotide to identify the base
4) Re-constructing the sequence
What are the steps of the reaction of dideoxy chain termination?
1) Sequencing reaction
2) Strand separation
3) Annealing primer
4) Extension
5) Chain Termination
Expand on the first 4 steps of the reaction of dideoxy chain termination.
• DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
• A single stranded oligonucleotide (primer) is bound to the template,
the polymerase recognizes the DNA structure, then forms an initiation complex
•Elongation from the 3’ terminus of the primer in a 5’ to 3 ‘ direction.
• Can also cool a denatured template to Tm of the primer to allow annealing.
RECAP: What does DNA dependent DNA polymerase require?
1) A template strand that extends beyond a primer
2) Free 3’ OH group on the primer
3) All 4 Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
4) Mg2+ ions (its a cofactor)
What happens at the elongating strand in addition of dNTP?
- Phosphodiester bond between the phosphate on the 5th carbon and the OH group on the 3rd carbon.
- Release hydrogen ions and inorganic pyrophosphate. (Small amount of hydrogen ions produced compared to in PCR)
What are the requirements of chain termination?
1) A template strand that extends beyond a primer
2) Free 3’ OH group on the primer
3) All 4 Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
4) All 4 Dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP) ~ Each one labelled with different fluorescent colour to you can tell which one is present
5) Mg2+ ions (its a cofactor)
How an elongating DNA strand terminated?
- Phosphodiester bond between the phosphate on the 5th carbon and the OH group on the 3rd carbon
- Release hydrogen ions and inorganic pyrophosphate. (Small amount of hydrogen ions produced compared to in PCR)
- Missing hydroxyl group on the dideoxy prevents further extension - as DNA polymerase will not be able to add more nucleotide to the elongating strand
• The particular dideoxy nucleotide added as a specific fluorescent colour attached to it, so we can tell which one has been added.
Why is the different fluorescent colours important for sequencing?
- 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.
- Grouping the different colours and then ordering these molecules by size allows us to determine the sequence of the new strand
Explain how the products of the reaction are separated.
- 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
• Gel electrophoresis is done in a capillary (a fine tube) filled with matrix
nucleotides are attached.
What happens when the sample passes the detector? and how is the sequence determined?
- The different molecules give out fluorescent at different wavelengths and the amount of light increases when they pass the detector. - when they pass through, the peak falls to baseline ~ there is a generation of an electropherogram (a trace)- and capture the light that is emitted and so you can identify which of the 4
- Measurement of fluorescence generates a trace and base calling is automated
- The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxy-nucleotides.
How is a sequence automated?
- By ABI 3730
- Samples prepared by dideoxy chain termination on a large scale by robotics
- Has read the length of up to 900bp and 99.95% accuracy
- Handles 48 or 96 samples simultaneously
- More than 1000 samples per day
- Only performs the separation of labelled DNA and determines the sequence – requires considerable hands on manipulation
- This technique was used to sequence the human genomes
How is DNA sequencing by dideoxy chain termination used in health?
- Still used, as it is still the gold standard confirmatory test for specific genetic mutations in patients with suspected genetic diseases.
- Used to confirm all types of mutation (Silent, Misense, Nonsense, Truncating, Indel, and Mis-Splicing) except low frequency mosaicism
- Identifies HIV haplotypes resistant to anti retrovirals HAART (highly active antiretroviral therapy) - if treatment will fail or not, if so, you need to modify this.
How is DNA sequencing by dideoxy chain termination used in research?
- Mammalian and pathogen gene sequencing
- Clone or PCR amplicon sequencing to confirm a cloned or site directed mutagenesis
- Walking a gene to identify a causative mutation in candidate gene studies
- Confirmation of causative variants associated with genetic disease following association study
