week 4

Question 1

describe sanger DNA sequencing

Answer 1

• sanger DNA sequencing was the dominant sequencing method from the 1980s to around 2010
• it is still widely used because it is easy to setup and very accurate for short regions (up to ~500 bases)
• the principal of the sanger method - replication of a DNA sequence in vitro using a DNA polymerase, dNTPs and dideoxy chain terminators (ddNTPs)

Question 2

what are dideoxy chain terminators

Answer 2

• dideoxy chain terminators are identical to normal dNTPs except that the -OH is missing from the 3’ position of the deoxyribose part
• the 3’ -OH is necessary for the subsequent addition of dNTPs to a growing DNA molecule
• thus, if a dideoxy chain terminator is incorporated into a growing DNA molecule, that molecule will grow no more

Question 3

describe manual sanger sequencing

Answer 3

• starts with single-stranded DNA template that you want to sequence
• a short, radioactively labelled primer of known sequence is designed to anneal to the target DNA region
• set up 4 separate reaction tubes, each containing:
  
  • the DNA template
  • the primer
  • DNA polymerase
  • all 4 dNTPs
  • a small amount of one specific ddNTP
    in each tube
• DNA polymerase builds new strand from primer, incorporating dNTPs
• occasionally, a ddNTP is incorporated, terminating the chain
• this results in a mixture of DNA fragments of varying lengths, all ending at a specific base
• all 4 tubes are then loaded into 4 adjacent lanes on a polyacrylamide gel and the fragments are separated by size using gel electrophoresis
• the gel is then dried and exposed to x-ray film, producing dark bands when DNA fragments are located
• each lane corresponds to a specific base
• the sequence is read manually from the bottom (smallest fragment) to the top (largest fragment)
• by reading the bands across the four lanes in order, you can reconstruct the complementary DNA sequence of the template strand

Question 4

describe automated sanger sequencing

Answer 4

• only one tube required containing:
  
  • DNA template
  • primer
  • DNA polymerase
  • all 4 dNTPs
  • all 4 fluorescently labelled ddNTPs
• fluorescent dyes are used which are safer and more stable than radioactive labels
• the mixture then goes through thermal cycling i.e. denaturation at 95C to separate the strands, annealing at 55C for primer to bind to template, extension at 72C for DNA polymerase to build strand until it randomly incorporates a ddNTP -> termination
• produces a collection of DNA fragments of varying lengths each ending with a fluorescently labelled base
• the mixture is then loaded into a capillary electrophoresis machine with high voltage current - short fragments are faster than long
• a laser is used to detect the fluorescent dyes attached to the terminal ddNTPs
• each colour represents a different base and a chromatogram is produced

Question 5

common uses of sanger sequencing

Answer 5

• confirming DNA edits/mutations
• sequencing cloned DNA

Question 6

what are other considerations for sanger sequencing

Answer 6

• Preparation of a DNA sample for sequencing. Millions of copies of the template DNA are required
  – It’s normal to use PCR to generate template DNA, or clone the unknown sequence into a plasmid (see later lectures).
• Requirement for a primer to initiate the reaction. The primer is actually essential for the technique because:
  – the polymerase will only extend a pre-existing chain
  – it ensures that all the chain elongation reactions will start at the same point
• The polymerase - desirable properties including fidelity, progressiveness, thermostability.

Question 7

describe automated sequencing

Answer 7

• Same principles as manual method:
  – Require template DNA, primer, ddNTPs, dNTPs, DNA polymerase, means of separating reaction products by size (gel), need for reaction products detection method
• Differences from the manual method:
  – Usually use of thermal cycling to obtain more copies of reaction products.
  – Use of fluorescently labelled dideoxynucleotides so that only one reaction is necessary instead of four.
  – Separation using liquid gel in a capillary and automated readout from a laser scanner.

Question 8

describe sequencing primer

Answer 8

• Sanger sequencing requires a primer, and therefore some knowledge of the sequence
• Sequencing primers are the same as PCR primers except:
  – Only 1 is required
  – They are usually a bit shorter (~16 to 22 bases long)
• If we wish to sequence an unknown piece of DNA, we can clone the DNA into a plasmid and use a primer that binds to the plasmid near the cloning site.