Sanger DNA Sequencing

Question 1

When and by whom was Sanger sequencing invented?

Answer 1

• Sanger sequencing, also known as dideoxy DNA sequencing, was named after Frederick Sanger
• He pioneered this new technology during the mid 70’s.
• Fred Sanger & Walter Gilbert received the Nobel prize in 1980 for developing DNA sequencing.

Question 2

What are the principles of Sanger DNA sequencing?

Answer 2

1. Sanger sequencing relies on random inhibition of chain elongation, creating newly synthesised fragments of various lengths that can be separated by size
2. Named dideoxy sequencing because of the involvement of 2’, 3’ dideoxynucleoside triphosphates (ddNTPs).
3. ddNTPs lack a hydroxyl group (OH) on the 2’and the 3’ of the deoxyribose sugar (fig 1).
4. The 5’C of ddNTP is able to form a phosphodiester bond with the previous nucleotide in the chain, but the 3’ C cannot form this bond with the next nucleotide (dNTP).
5. Without the 3’ OH group there is no place for the DNA polymerase to ligate the next nucleotide.
6. Addition of ddNTP terminates chain growth, this is the main principle to Sanger sequencing.

Question 3

What are input reagents required to perform Sanger sequencing?

Answer 3

1. DNA template
2. DNA primer
3. DNA polymerase (enzyme)
4. dNTPs
5. ddNTPs
6. Buffer

Question 4

What are the four main stages in a Sanger Sequencing reaction?

Answer 4

1. Strand separation
2. Primer annealing
3. Extension
4. Termination (KEY STEP)

Question 5

Describe the Strand Separation stage in a Sanger Sequencing reaction.

Answer 5

• dsDNA –> ssDNA
• This is accomplished by heating the dsDNA which breaks the hydrogen and Van der Waals bonds that are holding the two chains of DNA together in a double helix

Question 6

Describe the Primer Annealing stage in a Sanger Sequencing reaction.

Answer 6

• Small ssDNA (~20 bases) called an oligonucleotide anneals to the 3 ꞌ end of the template strand.
• To prevent the ssDNA from re-annealing a rapid cooling process occurs giving the primer sequence an advantage over the long DNA strand to anneal.
• Excess amounts of primers are used to ensure they will out-compete the complementary strand for annealing to the template.
• Primer annealing is required for the extension step to occur.

Question 7

Describe the Extension stage in a Sanger Sequencing reaction.

Answer 7

• Temperature is increased to allow DNA polymerase to function
• Starting at the primer, DNA polymerase uses the template strand as a guide adding complementary nucleotides to create a new DNA strand
• DNA polymerase makes no distinction between dNTPs and ddNTPs
• The concentration of ddNTP is set much lower than that of the counterpart dNTP, and thus there is competition between the two for inclusion at any given point.
• Since the dNTP is in excess, the majority of the time this will be incorporated and elongation will continue.

Question 8

Describe the Termination stage in a Sanger Sequencing reaction.

Answer 8

• Occasionally, the ddNTP will be incorporated, ending DNA synthesis and causing chain termination. Chain termination will occur randomly at one of the many different positions that will accept that specific base.
• This creates a collection of DNA fragments of different lengths, each with the same 5’ end (due to primer) but variable 3’ ends within each of the four separate reactions.

Question 9

How are the products of a Sanger sequencing reaction analysed to determine the sequence of the template DNA molecule?

Answer 9

Following sequencing a clean up step is needed to remove any unincorporated ddNTP’s, primers, non-specific DNA and excess salts.

The differently chain-terminated products can then be size-fractionated on a polyacrylamide gel or (as is commonplace nowadays) by c_apillary electrophoresis_

Question 10

How was size-separation of chain-terminated products performed in the early years of Sanger sequencing?

Answer 10

• In early Sanger assays four separate sequencing reactions are performed.
• Only one of the four dideoxynucleotides (ddATP,ddGTP,ddCTP or ddTTP) is added to each of the separate reaction mixtures.
• The four separate reactions were run out on separate wells on a polyacrylamide gel and visualised on an autoradiograph.
• The DNA sequence was read by reading the order of bands accross the four lanes from the top of the gel (larger fragments) to the bottom (small).

Question 11

How is size-separation of chain-terminated products commonly performed now?

Answer 11

• Automated DNA sequencing machines developed in the early 1990’s used fluorescent labelling of DNA in a safer reaction.
• Four different fluorescent dyes were used in the four base-specific reactions, meaning all four reactions be performed together using all four of the ddNTPs, each labelled with a different coloured fluorochromes
• Capillary electrophoresis enabled the migration of sequencing products through long, thin glass capillaries containing polyacrylamide gel
• A laser at a fixed point in the gel, which excites the fluorescently labelled ddNTPs. A monitor detects and records the wavelength to form an electropherogram of nucleotide sequence.

Question 12

What are the commonly used systems for performing capillary electrophoresis and analysing electropherogram traces?

Answer 12

• Applied Biosystems ABI capillary sequencers are most common
  
  • 3130 = 16 capillary injection
  • 3730 = 48 capillary injection
• Applied Biosystems BigDye sequencing kit consumables
• Mutation Surveyor software translates the raw data into the corresponding nucleotide bases, and also assigns a quality score to each base

Question 13

What are the advantages of Sanger DNA sequencing?

Answer 13

• Reads of 800-1000 bp can be generated using SS, major advantage of NGS.
• Can characterise large indels, repeat expansions which may be missed with NGS.
• Can handle highly homologous functional genes/pseudogenes better than NGS.
• SS is considerably less reliant on computational tools than NGS i.e. data processing stage is cheaper, storage is cheaper.
• Individual PCR’s can be optimised so that ‘hard to sequence’ regions are not lost, as with NGS.

Question 14

What are the disadvantages of Sanger DNA sequencing?

Answer 14

• Throughput much lower than NGS even when using 3730 ABI machines and robots
• Bioinformatic analysis constantly improves detections of difficult mutations such as indels etc especially from WGS
• Cost of NGS is dramatically decreasing to the point where WGS is becoming feasible, therefore Sanger is expensive in comparison
• SS requires a larger amount of template DNA than NGS
• SS requires often arduous and costly PCR optimisation prior to sequencing
• SS can affected by variants under primer sites and poly tracts in the template
• SS relies on peak height differences therefore is less sensitive than NGS in detecting low frequency variants