11.3: DNA Sequencing (Lilly's version) Flashcards
What are most methods of DNA sequencing based on?
DNA synthesis
what does DNA polymerase do?
catalyses the formation of bonds between phosphodiester bonds (water is a by product)
what is the Sanger-dideoxy chain terminating method?
- using dideoxyribonucleoside triphosphate to terminate DNA synthesis
- a) normal dNTP extends DNA strand
- b) dideoxy (ddNTP) terminates strand
consider 5% ddGTP
- 95% of the time when dGTP is incorporated It will elongate normally
- but 5% of the time ddGTP will be incorporated and terminate the sequence = results in DNA daughter strands of varying length depending on where that G occurs in the sequence
why would we spike a DNA polymerization cocktail with ddATP, ddCTP, ddGTP, and ddTTP? how could this be visualized?
- we could get a subset of DNA elongation products termination at every position in DNA sequence - determines sequence
- using fluorescent probes and gel electrophoresis we can visualize it
what type of gel does fluorescent dideoxy sequencing use? How is the visual captured?
- it uses polyacramide gel (contains urea) to separate ssDNA fragments by size
- gel provides very fine resolution, can tell difference by one base size
- as ddNTP-terminated fragments migrate through the gel, they pass a laser beam that excites fluorescent dyes and a camera that record the flash of light records those results
What is the problem with Sanger dideoxy sequencing?
- state of the art in the 70s
- accurate but SLOW
- og. 4/tube/ sequence
- now 100 samples / day but still slow
what are the pros of sanger dideoxy sequencing?
- accurate
- relatively long sequencing reads (up to 1000, normally ~650)
- low cost
- easy to do (can be automated)
What are the cons of sanger dideoxy sequencing?
- too slow for many applications
- costly when scaled up to aware lots of data
- requires purification and preparation of each individual
- limitations lead to next generation methods
the human genome project literally cost 3 billion dollars partially due to these drawbacks
What drove production of the ‘next generation’ methods?
- the inefficiencies of dideoxy sequencing methods
why didn’t automating the sanger process help?
- helped but it wasn’t enough: each sample needed to be purified to consist of only 1 DNA sequence
What was the solution to single sequencing being too slow?
- massively parallel sequencing instead!
- approach need that allowed for millions of DNA segments to be sequenced at once
What are the next generation sequencing methods?
- pyrosequencing
- illumina
- ion torrents
all use sequencing via DNA synthesis
What is illuminated DNA sequencing?
DNA samples attached to surface, form bridges and then the double stranded molecules of the bridge are denature into single stranded
- the first base is determined, then the second base, : multiple chemistry cycles, data aligned
What are the key points of illumina DNA sequencing?
- DNA fragments need to be short
- adaptor sequences must be added by ligation to the ends of the DNA sequences: helps the primer bind and the strand to attach to the oligonucleotides on the surface of the flow cell
- DNA segments are randomly arrayed across flowers cell sequence
What occurs after the DNA segments are attached to the flow cell surface?
- bridge amplification: amplify single DNA molecules into clusters of identical DNA molecules
How does sequencing in illumina sequencing occur?
sequencing occurs by addition of fluorescent nucleotide analogs 1 base at a time
- these dNTPS are chain terminators (like sanger) but are reversible so that elongation can continue
Why are the dNTPs terminators in illumina sequencing? How long does this process occur?
- after each dNTP is added the sequencer pauses and exposes the flow cell to a laser which takes picture to record was dNTP was added
- this process continues for a few hundred cycles
- the computer interprets the data; this way millions of sequences can be performed at a time = massively parallel!
what is 3rd generation sequencing? what is an example?
- nanopore sequencing
faster, single molecule, longer reads
What is Nanopore Sequencing ?
- NOT DNA sequencing by synthesis
- single molecule at a time (no PCR amplification required)
- enzyme unwinds DNA; a single strand is pulled through by an electrical current through a membrane
- each base produces characteristic disturbance in electrical current, used to read the base
Which forms of sequencing require DNA synthesis?
- first generation and second generation
- they also require amplification of those individual strands
What are the pros of nanopore sequencing?
- long reads: up to 100kb
- no amplification step
- can be used in the field to get rapid results
- can detect methylated bases
what are the cons of nanopore sequencing?
- slightly less accurate than other methods
Evaluate the three generations on whether they are: massively parallel, sequenced by synthesis, a single molecule (ie: not prepped), a chain terminator, the accuracy, and the read length
Sanger dideoxy chain terminator: not massively parallel, sequenced by synthesis, not a single molecule (a fragment that is amplified), it is a chain terminator, highly accurate (99.99%) and relatively short read length (65-1000)
- second generation: illumina: massively parallel, sequenced by synthesis, not a single molecule (short segments), a reversible chain terminator (pauses and then keeps going), 99.9% accuracy, read length (75-600 bases)
- third generation: nanopore: massively parallel, not sequenced by synthesis, a single molecule (no prep), not a chain terminator, lower accuracy (98-99%) and read length >100kb
does NGS have a greater read length or sanger?
technically illumina evaluates less bases at a time, BUT is faster so overall has a greater output
