DNA Sequencing Flashcards
What is the general process of the Sanger Method?
There is a use of a primer to sequence a new chain, you will have an H instead of the OH on the sugar which forms the 5’ link with the phosphate. Adding ddNTPs will cause the termination of the sequence and depending on how much you have, it will stop at different points. You then use polyacrylamide gel with urea to discriminaye ssDNA differing in length by one nucleotide and test through autoradiograph.
What is the ideal ratio for dNTPs:ddNTP and why is the ratio important?
The ideal ratio is 5:1 and the reason for this is that it will allow for the sequence to extend longer, if there was a 1:1 ratio there is a 50% of the reactions will stop with the first match it comes acoss
How does fluorescent labelling improve the Sanger method?
4 ddNTPs are labelled with different colours for easier reading and you can run the 4 reactions in one tube and analyze the products in one lane
How do thermostable polymerases better the Sanger method?
There is a high rate of processivity and no exonuclease activity because 5-3’ will discriminate against ddNTPs and 3–5’ will remove primers
How does the use of Capillary electrophoresis better the Sanger method?
Load several samples more tightly into a capillary and has laser sensor and will take place right after the reaction
How does ABI DNA sequencer better the Sanger method?
The bases will be labeled with fluorophores and each label has an absorbance and emission spectra. Two filters are placed over the samples, one that measures the absorbance and one that measures the emission. You can only have one primer because you will not have clear peaks
What are the general steps for NGS?
- Preparing the library
breaks DNA into shorter
fragments
attach adaptors to the
ends
Run emulsion PCR or bridge PCR to amplify from single template
What are the general steps for pyrosequencing?
- Prepare library
- Run emulsion PCR
- Load reaction onto picoplate, and centrifuge
- put into a machine with substrate for reaction (ATCG) and wash, and repeat
How does pyrosequencing actually perform sequencing?
Pyrophosphate is released as DNA nucleotides are added, combined with APS produces ATP. ATP combined with luciferin and luciferase to produce a light. You add A, and if no light it produced you wash away. (repeat for other nucleotides) If you have larger peaks that is how much pyrophosphate produced
What are the steps of Illumina sequencing?
Prepare libraries of DNA fragments linked to adaptors, and attach to solid phase, undergo amplification by bridge PCR and undgero sequence by synthesis
What is meant by sequencing by synthesis?
Clustered clonal DNA fragments are anchored to solid phase and used as templates
in each cycle, only one base carrying a unique fluorescent label is added and carries a marker that blocks the 3’ OH, image is taken to identify the nucleotide incorporated, then block is removed to continue the sequencing
What are the general steps of the Ion Semiconductor Sequencing
DNA fragments are generated and adaptors are added, undergo emulsion PCR, one nucleotide is added in each cycle, if dNTP is added an H+ ion will be released meaning that there will be a change in pH, sensing change in pH will mean it is the correct base
What are the general steps of Pacific Biosciences Real Time Sequencing?
DNA polymerase phi29 is attached to bottom of a well through streptavidin and biotin bond. Bottom of the well is coated with silane PEG Biotin. The template is attached to the polymerase, fluorescently labelled nucleotides will bind and release a light if it’s the right base pair, and the light eventually goes away and the template translocates to prepare for the next binding. If there is an extended gap time between the binding you can tell if there is methylation or any other DNA modifications.
What is the general process of nanopore sequencing?
double stranded DNA with attached motor protein attaches to a pore protein with an insulating membrane, the applied potential unzips the DNA in a step wise fashion, once unzipped completely the complex detaches from the pore entrance. Translocation of DNA strand results in different conductance which is how nucleotides are measured.
What are the three biological based pores used in sequencing?
a-hemolysin, MspA and Phi29
What are the three non-biological based pores used in nanopore sequencing?
silicon based, Al2O3 and single layer membrane
