10- DNA Sanger Sequencing

Question 1

what is Sanger/dideoxy sequencing?

Answer 1

multi-stage DNA sequencing technique - allows us to determine the nucleotide sequence of a DNA fragment

Question 2

describe the process of dideoxy sequencing

Answer 2

1. a DNA template is produced by a PCR reaction with the region of interest
2. enzymatic sequencing/ sanger sequencing reaction using DNA dependent DNA polymerase to synthesise complementary strands to the template

occurs in four stages:
strand separation of the template DNA strands
annealing a primer complementary to the 5’ end of the template
extension of the primer by DNA polymerase using dNTPs
chain termination with random incorporation of labelled ddNTPs

enzymatic reaction occurs in cycles until enough differently sized DNA fragments have been synthesised

3. size separation of the differently sized DNA fragments by capillary gel electrophoresis
   - negatively charged nucleic acids are passed through a gel matrix in a capillary
   - larger molecules move slower
4. base calling
   - detecting reaction products using the fluorescently labelled ddNTPs involved in chain termination, different colours for the different types
   - determine sequence by analysing the fluorescence signals at each different position
   - electropherogram peaks produced by different fluorescence signals - characteristics of peaks used to identify the base associated with each peak
   - read the sequence of bases from base calling

Question 3

summarise the stages for the multi-stage process of sanger sequencing

Answer 3

1. template production by PCR for the region of interest
2. enzymatic sequencing reaction with DNA dependent DNA polymerase - involves strand separation, annealing the complementary 5’ end primer, extension of primer by DNA polymerase and random chain termination with fluorescently labelled ddNTPs
3. size separation of DNA fragments by capillary gel electrophoresis, where larger molecules move slower
4. base calling for sequence reading with an electropherogram where the different fluorescent signals for the different ddNTPs are represented by peaks, and peak characteristics can be used to identify the base at each sequence

Question 4

describe similarities between Sanger sequencing and PCR

Answer 4

similarities
- both use DNA dependent DNA polymerase = for enzymatic sequencing with Sanger sequencing, for DNA amplification with PCR

• temperature cycling = repeat cycles of temperature changes for denaturation, primer annealing and extension
• thermostable enzyme used due to temperature cycling

Question 5

describe differences between Sanger sequencing and PCR

Answer 5

PCR:
- exponentially amplifies a specific DNA target = limitless amplification until dNTPs run out
- product is used as a template for the next reaction
- two oppositely annealed primers (5’ overhang and free 3’ OH)
- selectively amplifies a specific region

Sanger sequencing
- doesn’t involve exponential amplification
- product isn’t used as a subsequent template
- single forward 5’ primer
- determines nucleotide sequence of a DNA fragment

Question 6

describe healthcare applications of dideoxy sequencing

Answer 6

• confirms different types of (specific) monogenic disease-causing mutations in patients = e.g. indels, missense or nonsense, truncating
• determining HIV haplotype in patients to determine HAART treatment

Question 7

describe research applications of dideoxy sequencing

Answer 7

• sequence genes in mammalian cells and pathogens
• confirm the sequence of clones or PCR amplicons = important for genetic research
• ‘walking’ a gene in candidate gene studies = sequencing the gene in segments to identify potential causative variants for a disease