18.02.13 Sanger sequencing

Question 1

What are the principles of Sanger sequencing?

Answer 1

1. Named dideoxy sequencing because of the involvement of 2’, 3’ dideoxynucleoside triphosphates (ddNTPs).
2. ddNTPs lack a hydroxyl group (OH) on the 2’and the 3’ of the deoxyribose sugar (fig 1).
3. 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).
4. Without the 3’ OH group there is no place for the DNA polymerase to ligate the next nucleotide.
5. Addition of ddNTP terminates chain growth, this is the main principle to Sanger sequencing.

Question 2

What stages occur prior to the sequencing reaction?

Answer 2

Prior to the sequencing reaction DNA is extracted and the target DNA is amplified via polymerase chain reaction (PCR). This creates many identical copies of the region being sequenced. Primers with a common tag are typically used for the initial template-producing PCR, meaning that all fragments can be subsequently sequenced using the same sequencing primers that are specific for the tag sequence.

Question 3

What is required in a sequencing reaction?

Answer 3

DNA sequence of interest to act as a template

DNA primer- a short piece of DNA complementary to the DNA you want to sequence

DNA polymerase (enzyme)

dNTPs

ddNTPs

Question 4

What are the four main steps to the sequencing reaction?

Answer 4

1. Strand separation (dsDNA -> ssDNA) by heating to break the H bonds of the double helix.
2. Primer annealing (ssDNA, 20nt) anneals to 3’ of template. Rapid cooling prevents ssDNA from re-annealing. Excess primer outcompetes the complementary strand for annealing.
3. Extension: temperatures is increased to allow DNA polymerase to function. DNA polymerase uses the template strand as a guide adding complementary nucleotides to create a new DNA strand making 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.

4. Termination (KEY STEP)
   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.

These differently sized fragments can be size-fractionated on a polyacrylamide gel or (as is commonplace nowadays) by capillary electrophoresis.

Question 5

What is required after sequencing, prior to electrophoresis?

Answer 5

Following sequencing, before electrophoresis, a second clean up step is needed to remove any unincorporated ddNTP’s, primers, non-specific DNA and excess salts. This can be done using ethanol precipitation or commercial kits.

Question 6

What is involved in capillary sequencing?

Answer 6

Capillary sequencing involves the migration of sequencing products through long, thin glass capillaries containing polyacrylamide gel with the smaller fragments moving quicker past a laser at a fixed point in the gel, which excites the fluorescently labelled ddNTPs. A monitor detects and records the wavelength of the specific bases creating a trace in the form of an electropherogram.

Question 7

What are some applications of Sanger?

Answer 7

Gene screen
Gapfill
Familial variant studies
NGS confirmations

Question 8

What size reads can be generated through Sanger sequencing?

Answer 8

Reads of 800-1000 bp can be generated using SS.

The shorter reads (40-400 bp) generated by second generation sequencing platforms mean that these platforms are more limited in their ability to resolve repetitive regions (eg: they would not be able to determine the actual number of repeated glutamine residues coded by the Huntingtin gene because the read lengths obtained are shorter than the repeated stretch of coding DNA). However, the parallel nature of these technologies means that longer reads of non-repetitive regions can be constructed from many contiguous short reads.

Question 9

What are some of the advantages of Sanger sequencing?

Answer 9

1. Less reliant on computational tools than NGS for alignment and analysis.
2. Easier to detect indels and align reads in regions containing highly homologous psuedogenes.
3. Uses up less data space than NGS data

Question 10

What factors will influence the quality of Sanger sequencing?

Answer 10

1. Initial template-generating PCR
2. Primer specificity
3. Positioning of primers around ROIs