L3 DNA sequencing

Question 1

average PCR process, selection

Answer 1

• add in primer
• DNA fragments amplified and then put into plasmids
• plasmids transformed into E. coli which is then plated onto agar plates
• agar plate contains ampicillin or other antibiotics
  => plasmid also contains resistance gene which E. coli doesn’t have
  ==> only E. coli with plasmid can grow
• DNA sequence to find correct plasmid

Question 2

what’s on a plasmid?

Answer 2

• promoter… gene… terminator
• ORI so it can be replicated
• bla = beta-lactamase which gives resistance to Ampicillin etc
• cat = gives resistance to other antibiotics
  => the last 3 are “backbone” of plasmid

Question 3

sanger sequencing

Answer 3

= sequencing by doing PCR to one small region

Question 4

sanger sequencing process

Answer 4

• add primer to template
• Pol will pull in dNTPs corresponding to the corresponding base (dCTP for G etc.)
• it will also randomly pull in ddNTPs corresponding to the base
  -> terminating nucleotides since they don’t have extra OH-group => no other nt can bind to it
• this leads to having sequences of all sizes terminated at random, one nt further than the other and so on
  => can separate them by size

Question 5

sanger sequencing seperation

Answer 5

• put DNA into electrical field
  => negatively charged DNA will go towards positive charge and shorter strands will move faster
• ddNTPs glow in different colour and a camera will then see which ddNTP is at the end as the sequence goes past it
  => plot will be created where you can see a peak of the colour at a specific time

Question 6

limitations of sanger sequencing

Answer 6

• rare mutation will create a very small peak in the chromatogram which is easy to miss
  => not good for rare mutations
• limited to 1000 bp
  -> too few ddNTPs: chain will continue without stopping
  -> too many ddNTPs: chain will stop straight away
• not good for mixture of DNA since it will give overlapping tops in chromatogram

Question 7

benefits of sanger sequencing

Answer 7

• cheap => 30 kr for one reaction
• good when knowing what mutation to look for

Question 8

NGS, MPS

Answer 8

Next Generation Sequencing, Massively Parallell Sequencing

Question 9

NGS process

Answer 9

• put small fragments of DNA onto a surface so a camera can follow them
• put primer and DNAP to each strand so PCR reaction happens to everyone at the same time

Question 10

limitations of NGS

Answer 10

• expensive
• each strand can be maximum 200 nts, but 10^8 can happen at the same time so it makes up for it in a way

Question 11

benefits of NGS

Answer 11

• good for sequencing large DNA pieces, like a whole genome
• good for detecting a mutation from population
  -> strands seperated from each other so easy to see rare mutations
• can estimate number of mutant DNA in population
• can quantify gene expression by converting all mRNA to DNA with reverse transcriptase