Profiling sequence

Question 1

Troubleshooting

Primer dimer

Answer 1

• The primer defines the reaction process and can bind itself together.
• Primer can dimerize itself so we just get a reformation of the double helix.
• This can be caused by too high primer concentration.

Question 2

Profiling

Answer 2

• Looks at allele level differences
• Provides information about familiar relationships
• Useful in forensic science
• Relies on polymerase reactions

Question 3

Sequences

Answer 3

• Looks at single base level sequence
• Provides information about proteins expressed
• Useful for biomedical analysis
• Relise on polymerase reactions

Question 4

Sanger sequencing overview

Answer 4

• Deoxynucleoside 5’-triphosphates (dNTPs): DNA synthesis monomers
• Dideoxynucleoside 5’-triphosphates (ddNTPs): Terminates DNA synthesis. Taking off the OH group terminates the synthesis as there’s no group for it to bind to. This is useful as it gives us information at everypoint it stops. Repeat this process 4 times with the 4 different bases as ddNTPs

Question 5

Sanger sequencing

ddGTP

Answer 5

• This means we can identify where the G groups are from the base pairs
• We know the length in terms of residues.
• Every point where they wanted to put a G in we can identify from the length of the chain.
• Position of bands tells you which positions have G, since G is the final base in each of these.

Question 6

Visualising a sanger sequence

Answer 6

• ³²P labelled ddNTPs
• Fluorescent dyes

Question 7

VIsualising sanger sequence

32 P

Answer 7

• Radioactive phosphate 32 group.
• Effective but has issues from radioactivity activating it.
• It is hard to read the sequences though.
• have to run it 4 times / use 4 different lanes

Question 8

Visualising a sanger sequence

Fluorescent dyes

Answer 8

• Label each ddNTP with a different colour of fluorescent dye.
• Run one PCR & one lane in a gel.
• Read fluorescent intensity to obtain the sequence.

Question 9

Visualising a sanger sequence

Capillary electrophoresis

Answer 9

Use automated capillary electrophoresis and measure many samples at once.

Question 10

Next generation sequencing

Answer 10

• We fragment our DNA through vibration (ultrasound)
• each of these fragments are stuck to different material (bead, droplet or surface of a material)
• On the surfaces we use PCR to amplify them to get multiplied repeats attached to the surface.
• Once we have the separated fragments (still attached to the surface) we add one base pair at a time.

Question 11

Sanger sequencing performance

Answer 11

• Near perfect ability to accurarely replicate a template.
• One read per run (parallelisation)
• Length of build is limited to upto 900 bases (length of DNA read)
• Time per 10⁶ bases: ~400 hours
• Cost per 10⁶ bases: $2400

Question 12

Next-generation sequencing performance

Answer 12

• Fidelity (ability to accurately replicate a template): Excellent if multiple reads are combined
• Parallelisation (reads per run) upto 3 billion
• Length of DNA run (Limit of how long we can build): upto 15,000 bases
• Time per 10⁶ bases: as low as 0.1 seconds
• Cost per 10⁶ bases: as low as $0.1

Question 13

Nanopore sequencing

Answer 13

• Portable
• Simple
• Enzyme unwinds the double helix & passes a strand through a nanopore.
• A potential is applited as ions are in solution.
• The channel in the nanopore is just the right size for the DNA strand to pass through.
• Each DNA base is a different size, so block nanopore differently - measured by change in current/

Question 14

How does nanopore sequencing work?

Answer 14

• Enzyme unwindes the double helix
• A strand passes through the part and goes through a designer channel that’s the right size for DNA and not the noise.
• It’s the right width to fit the base pairs.
• Then it gets put into solution with salts and ions in, the flow of those is measured as a voltage.
• The movement of the ions in the two sides is measured and that relates to whatever base pair is present.
• Background levels will be a problem and need to be thought about
• There are less residues from G and T as they’re bulkier
• The drawback of this is it is quite expensive to perform