DNA sequencing Flashcards
How has DNA sequencing 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 with a 99.95% accuracy. It handles 48/96 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
AKA Sagner sequencing
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 are sorted by size, and as 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?
1) Generating a template
This can either be a clone (plasmid) or an amplicon (from PCR)
2) Sequencing reaction
DNA polymerase makes multiple copies of the template
3) Separation on size
This is done using capillary electrophoresis
4) Detection of reaction particles
Here, sequential detection of the terminating nucleotide to identify the base occurs
5) Readout of the sequence
This is where the sequence is reconstructed
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, dideoxy sequencing only uses a single forward primer, meaning amplification is limited and not expontential.
They both use a DNA polymerase
What are the different steps in the sequencing reaction in dideoxy chain termination?
1) Strand separation
2) Annealing primer
3) Extension
4) Chain termination
Expand on the first two steps of the sequencing reaction
The first two steps are strand separation and annealing primer.
The DNA is mixed with the reaction components and a labelled, single stranded nucleotide is bound to the template. The polymerase recognises the DNA and forms an initiation complex and commences elongation from the 3’ terminus of the primer in a 5’ to 3’ direction
Expand on the third step of the sequencing reaction
The third step is extension.
This occurs by the action of DNA polymerase and requires:
- a template strand that extends beyond a primer
- free 3’ OH group on the primer
- all 4 dNTPs
- Mg2+ ions
Expand on the fourth step of the sequencing reaction
The fourth step is chain termination.
DNA elongation is terminated by the addition of a dideoxynucleotide, which prevent elongation.
As the enzyme encounters a particular nucleotide 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
Explain the implications of adding the dideoxy molecules into the sequencing reaction
Products where a ddCTP is incorporated represent all positions within the sequence where a cytosine occurs. Since all four labelled dideoxy nulceotides are present in the reaction, the population of molecules produced represents all the possible reactions in the sequence from the same point to the end.
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 the 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
How is the sequence determined after it is size separated?
The sequence is determined simply by the direct comparison of the lengths of products terminated by each of the four dideoxynucleotides
What are the implications of DNA sequencing by dideoxy chain termination in health?
They are used to confirm almost all types of mutation:
- silent
- missense
- nonsense
- truncating
- indel
- missplicing
however, not low frequency mosaicism
they are also used in identifying HIV haplotypes that are resistant to anti-retrovirals HAART
What are the implications of DNA sequencing by dideoxy chain termination in research?
- mammalian and pathogen gene sequencing
- clone or PCR aplicon sequencing
- ‘walking’ a gene to identify a causative mutation in candidate gene studies
- confirmation of causative variants associated with genetic disease following association study
