19.02.13 Sanger sequencing

Question 1

What is Sanger sequencing used for?

Answer 1

• Mutation scanning and testing
• Developed in the 1970s
• Relies on random inhibition of chain elongation

Question 2

Principles of Sanger sequencing

Answer 2

• Uses dideoxynucleotide triphosphates (ddNTPs)
• ddNTPs lack a hydroxyl group (OH) on the 3’ carbon of the sugar ring
• This is needed to form the phosphodiester bond between one nucleotide and the next one during strand elongation
• DNA polymerase can’t ligate the next nucleotide, therefore inhibiting further strand extension
• The ddNTPs are labelled with a particular coloured fluorochrome (different colour for A, T, C and G)
• So this process produces DNA fragments of varying lengths, which all end in a labelled ddNTP

Question 3

Classical chain-termination method

Answer 3

• Amplify target DNA by PCR - primers with a common tag (m13) are used to allow a common sequencing primer to be used later
• PCR products run on gel to check for contamination and check amount of PCR product
• Clean up PCR product to remove unincorporated primers and dNTPs
• Sequence DNA in a reaction containing PCR product, primers, DNA polymerase, dNTPs and all 4 ddNTPs(at a low conc))
• Sequencing has 4 stages
  1) Strand separation - heat to denature dsDNA to ssDNA
  2) Primer annealing - Cooled so primer can bind to ssDNA template (Rapid cooling to favour primer binding, rather than ddDNA formation)
  3) Extension - Temp increased to let DNA polymerase work on template strand. It starts at primer and adds complementary nucleotides to create new DNA strands. Conc of ddNTPs is lower than dNTPs, so they are rarely added and elongation continues
  4) Termination - ddNTP is incorporated - due to lack of 3’ OH group in sugar ring that terminates elongation and get chain termination. Occurs randomly at one of the many different positions. Creates collection of DNA fragemtns of different lengths, with the same 5’ end (due to primer) but variable 3’ ends.
• Following sequencing, get a second clean up step
• Then separate different sized fragments by capillary electrophoresis

Question 4

Capillary sequencing

Answer 4

• involves the migration of sequencing products through long, thin glass capillaries
• Smaller fragments move faster past laser
• Laser excites fluorescently labelled ddNTPs
• Monitor detects wavelength emitted and records base at that position

Question 5

Sanger data analysis

Answer 5

• Commonly completed using Mutation Surveyor

- Translates raw data into nucleotide base and assignes a quality score (i.e. how confident of no base call error)

Question 6

Clinical applications and limitations

Answer 6

• NGS is now very popular and great for large panels
• Sanger is still gold standard for confirming NGS findings, as well as testing for known familial mutations and mutation hotspots. Also can be used for gap fills in NGS coverage
• Sanger can create reads of 800-1000bp - which is good for repetitive regions where NGS struggles
• Sanger need less bioinformatic input, compared to NGS, for aligning and analysing data
• Sanger needs more template DNA than NGS
• Positioning of primers is important - variants under binding site can cause allele drop out, and variants close to primer can cause poor sequence quality
• Can’t detect low level mosaicism, which could be seen by NGS.