Profiling and Sequencing

Question 1

What are the steps in DNA profiling?

Answer 1

1. 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

Question 2

What is profiling vs sequencing?
- looks at?
- provides?
- useful for?
- relies on?

Answer 2

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

Question 3

What are ddNTPs?
- name
- structure
- use
- compared to dNTPs

Answer 3

• 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

Question 4

What is sanger sequencing?

Answer 4

• 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)

Question 5

How can read a Sanger sequence?

Answer 5

• 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

Question 6

What is an easier way to visualise a Sanger Sequence?

What could be used but has disadvantages?

What can even be done nowadays?

Answer 6

• 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

Question 7

What is instrumentation like for sanger sequencing?

Answer 7

• automated box with liquids

Question 8

What is NGS
- name

Answer 8

• 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

Question 9

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

Answer 9

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

Question 10

Nanopore sequencing:
- process
- two pros
- a con

Answer 10

• 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

Question 11

what modification of dNTPs are needed for Sanger sequencing? [3 marks]

Answer 11

• 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.