DNA Sequencing (OCR A Level Biology)

• DNA sequencing allows the nucleotide base sequence of an organism's genetic material to be identified
• 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

Sanger sequencing

• The chain termination method of DNA sequencing uses modified nucleotides called dideoxynucleotides
  • Dideoxynucleotides have a slightly different structure to the 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 why 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, producing a shortened DNA chain

The chain termination method in action

1. 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 *
2. The test tubes are incubated at a temperature that allows the DNA polymerase to function
3. 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
4. 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
5. 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)
6. 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
7. Once the incubation period has ended the new, complementary, DNA chains (also referred to as the developing strands) are separated from the template DNA
8. 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 is a term that describes multiple DNA sequencing technologies, all of which allow simultaneous sequencing of multiple DNA strands
  • High throughput methods are rapid and so produce large datasets very quickly
• Some high-throughput sequencing methods employ a faster version of the chain-termination technique, e.g. capillary gel electrophoresis, which involves the following:
  • each type of dideoxynucleotide is labelled using a specific fluorescent dye
  • the single-stranded DNA chains are separated according to mass using electrophoresis that is carried out inside a capillary tube
  • 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 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 increase in speed enabled by high-throughput sequencing has allowed scientists to sequence and analyse the genomes of many organisms; this is useful for fields of study such as:
  • evolutionary biology and classification
  • personalised medicine
  • disease diagnosis