Profiling and Sequencing Flashcards
What are the steps in DNA profiling?
- purify the sample and extract DNA (extract from cells and separates from cellular components)
2 - quantify the amount of DNA to make sure there is enough
3 - use PCR to make copies of DNA
4 - STR analysis of DNA fragments - use gel electrophoresis to separate them
What is profiling vs sequencing?
- looks at?
- provides?
- useful for?
- relies on?
profiling
- looks at allele-level differences (subsection of gene/gene level differences)
- provides info about familial relationships
- useful in forensic science
- relies on polymerase reactions
sequencing
- looks at single base-level sequence (looks at every base and gives more resolution)
- provides info about proteins expressed
- useful for biomedical analysis
- relies on polymerase reactions
What are ddNTPs?
- name
- structure
- use
- compared to dNTPs
- dideoxynucleoside 5’-triphosphates
- OH group on 3’ removed
- they terminate DNA synthesis - no OH group so cannot function
- dNTPs are building blocks of base pairs attached to sugars attached to triphosphate groups
- dNTPs have OH group on 3’ which allows bridging between OH group on sugar group and next base pair via the phosphate
- dNTPs are synthesis monomers
- whereas ddNTPs have OH group on 3’ removed so is end of DNA synthesis
What is sanger sequencing?
- primer gets added
- DNA polymerase adds on dNTPs to replicate the DNA sequence and ddGTPs which stops polymerisation
- have mixture of dNTPs and ddGTP to produce a variety of different lengths
- using primer can identify exact position relative to our primer of exactly where G groups are
- once got all possible lengths, for each identify ddGTP that stopped polymerisation
- can measure length by position of bands on PAGE gel (this tells you which positions have G since G is final base in each of these)
- shorter fragments travel further through gel than longer ones so at bottom of gel
- repeat same process but test the sample 3 more times with other ddNTPs (C/A/T)
How can read a Sanger sequence?
- if run all 4 ddNTPs then get full picture of entire structure we have
- read from top to bottom to get complementary sequence of original DNA strand (3’ to 5’)
- if want original base pairs for original DNA strand, invert this and find base pairs
What is an easier way to visualise a Sanger Sequence?
What could be used but has disadvantages?
What can even be done nowadays?
- with all 4 ddNTPs in there at same time
- label each ddNTP with a different fluorescent dye
- all colours in single ladder
- read based on fluorescent intensity
- in a single run get a entire genetic sequence
- could label with 32P as very effective but has some issues as get radiation impacting it
- nowadays done with automated capillary electrophoresis and measure many samples at once.
- run through capillary and detect when things are moving past
- allows us to multiplex even more
What is instrumentation like for sanger sequencing?
- automated box with liquids
What is NGS
- name
- next generation sequencing
- fragment genomic DNA using ultrasound to vibrate it (break it up)
- isolate individual fragments and put on a different material (bead, droplet, surface arrays)
- amplify it using PCR to get multiple repeats
- add one base pair at a time and record which one is being added by electrical colour change to determine the sequence of fragments
- then use data reconstruction to get full sequence
Sanger sequencing vs NGS
- fidelity
- parallelisation (reads per run)
- length of DNA read
- time per 10^6 bases
- cost per 10^6 bases
which is better
sanger sequencing:
- near perfect fidelity
- one read per run
- up to 900 bases can be read
- around 400 hours for a million bases
- $2400 cost per million bases
NGS:
- excellent if multiple reads combined
- up to three billion reads per run (as have lots of different beads with different materials on it)
- up to 15,000 bases that can be read
- 0.1 seconds for a million bases
- $0.10 cost per million bases
NGS better but still emerging
Nanopore sequencing:
- process
- two pros
- a con
- enzyme unwinds the double helix and passes a strand through a nanopore
- the channel in the nanopore is just the right size for the DNA strand to pass through
- a potential is applied to ions in solution
- each DNA is a different size so block nanopore differently
- this is measured by change in current (amount of current that flows is based on bulkiness of base)
- portable
- simple
- expensive as still emerging
what modification of dNTPs are needed for Sanger sequencing? [3 marks]
- make ddNTPs instead of dNTPs
- removal of OH at 3’ position to terminate replication at specific base (need to be able to terminate and stop polymerase and take out OH groups – won’t be able to bridge to phosphate group)
- tagged with fluorophore or radioactive labels in order to enable visualisation of where the termination occurred using gel electrophoresis.
