6.3.1 DNA Sequencing

• DNA sequencing allows for the nucleotide base sequence of an organism's genetic material to be identified and recorded
• In the 1970s the chain termination method of sequencing was developed by Frederick Sanger and his colleagues
  • The chain termination method is also known as Sanger sequencing

• Advances in technology have enabled the development of high-throughput sequencing methods which allow scientists to rapidly sequence the genomes of organisms
  • The use of a method called capillary electrophoresis enables the chain termination method to be carried out in a high-throughput way (see below)
  • The newest high-throughput methods do not involve electrophoresis and are known as next-generation sequencing methods e.g. nanopore sequencing and pyrosequencing

• Most sequencing methods used are now automated rather than requiring manual interpretation
• The data obtained from sequencing can be entered into computers with specialised programmes that compare the base sequences of different organisms

The chain termination method, or Sanger sequencing

• The chain termination method of DNA sequencing uses modified nucleotides called dideoxynucleotides
  • Dideoxynucleotides have a slightly different structure to the nucleotides (which can be referred to as deoxynucleotides) found within the DNA of organisms

• Dideoxynucleotides can pair with nucleotides on the template strand during DNA replication
  • They will pair with nucleotides that have a complementary base

• When DNA polymerase encounters a dideoxynucleotide on the developing strand it stops replicating, hence this method of sequencing is referred to as the chain termination method

Once the dideoxynucleotide is added to the developing strand DNA polymerase stops the replication of the developing DNA strand to produce a shortened DNA chain

The chain termination method in action

• Four test tubes are prepared that contain the DNA to be sequenced (in the form of a single-stranded template), DNA polymerase, DNA primers, free nucleotides A, C, T, and G, and one of the four types of dideoxynucleotide; either A*, C*, T*, or G* (you may notice that this process bears a strong resemblance to PCR, but with the addition of dideoxynucleotides, which are notated here with *)
• The test tubes are incubated at a temperature that allows the DNA polymerase to function
• The primer anneals to the start of the single stranded template, producing a short section of double stranded DNA at the start of the sequence
• DNA polymerase attaches to this double stranded section and begins DNA replication using the free nucleotides in the test tube
  • Hydrogen bonds form between the complementary bases on the nucleotides

• At any time, DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of DNA replication
  • Because each of the test tubes only contains one type of dideoxynucleotide, it is possible to know what the terminal nucleotide of each fragment is (i.e. if the test tube contains A*, then researchers will know that the final nucleotide of every chain in that test tube is A)

• Because the point at which the dideoxynucleotide is inserted varies with every strand, complementary DNA chains of varying lengths are produced
  • These chains can vary in length from one nucleotide to several hundred nucleotides

• Once the incubation period has ended the new, complementary, DNA chains (also referred to as the developing strands) are separated from the template DNA
• The resulting single-stranded DNA chains are then separated according to length using gel electrophoresis
  • The gel will have four wells, one each for A*, C*, T*, and G*
  • A fragment that consists of only one nucleotide will travel all the way to the bottom of the gel, and every band above this on the gel represents the addition of one more base. E.g. If the band on the gel that travels furthest comes from the C* well, scientists can see that the first base in the sequence is C. If the next furthest band comes from the T* well, the second base in the sequence is T, and so on
  • This allows the base sequence to be built up one base at a time

High-throughput sequencing

• High-throughput sequencing can also employ the chain-termination technique by using a different method of fragment separation
• Each type of dideoxynucleotide is labelled using a specific fluorescent dye
  • Dideoxynucleotides with an adenine base (ddNA) are labelled green
  • Dideoxynucleotides with a thymine base (ddNT) are labelled red
  • Dideoxynucleotides with a cytosine base (ddNC) are labelled blue
  • Dideoxynucleotides with a guanine base (ddNG) are labelled yellow

• The single-stranded DNA chains are separated according to mass using a specific type of electrophoresis that takes place inside a capillary tube
  • Known as capillary electrophoresis 
  • This type of electrophoresis has a very high resolution. It is capable of separating chains of DNA that vary by only one nucleotide in length

• A laser beam is used to illuminate all of the dideoxynucleotides, and a detector then reads the colour and position of each fluorescence
• The detector feeds the information into a computer where it is stored or printed out for analysis

The process of capillary electrophoresis. This is a high-throughput way of carrying out the chain termination method. Note that because this method is essentially still Sanger sequencing, it cannot be referred to as next-generation sequencing

• The increase in speed enabled by high-throughput sequencing has allowed scientists to sequence and analyse the genomes of many organisms
• Scientists can determine the function of sections of DNA by 'knocking out' genes to see how this affects an organism
• Genes can be rewritten to alter their function, and then inserted into cells using genetic engineering techniques; this means that scientists can potentially design new molecules with huge potential for drug production (this branch of biology is known as synthetic biology)
• Genome sequence data can also provide information about evolutionary relationships

Next-generation sequencing

• Any method of DNA sequencing that has replaced the Sanger method is referred to as next-generation sequencing (NGS)
• Thousands to millions of DNA molecules can be sequenced at the same time (in parallel)
• NGS methods can be one thousand times faster than older methods of sequencing
• The reduction in time required for sequencing means that costs are also greatly reduced
  • NGS methods cost roughly 0.1% of the cost of chain-termination methods

• Nanopore sequencing is currently being developed by scientists
  • This method of sequencing will be extremely rapid and allow for sequence data to be obtained outside the lab and used for a range of applications

A vertical section through a nanopore, showing how it can be used in next-generation DNA sequencing