8.3.4 Sequencing Methods

• DNA sequencing allows for the base sequence of an organism's genetic material to be identified and recorded
• Sequencing methods are continuously evolving and becoming faster. Advances in technology have allowed scientists to rapidly sequence the genomes of organisms
• Most sequencing methods used are now automated
• The data obtained from sequencing can be entered into computers with specialised programmes that compare the base sequences of different organisms

DNA sequencing

• All methods of DNA sequencing use dideoxyribose nucleotides
• A dideoxyribose molecule is very similar in structure to ribose molecules and deoxyribose molecules
  • It has one less oxygen atom than a deoxyribose molecule and two fewer oxygen atoms than a ribose molecule

The dideoxyribose molecule has a very similar structure to the other molecules although it does contain fewer oxygen atoms

• Dideoxyribose can form nucleotides in the same way that ribose and deoxyribose molecules do, by binding to a phosphate molecule and an organic base
• Dideoxyribose nucleotides can pair with deoxyribose nucleotides on the template strand during DNA replication
  • They will pair with nucleotides that have a complementary base

• When DNA polymerase encounters a dideoxyribose nucleotide on the developing strand it stops replicating. This is the chain-termination technique that is used for DNA sequencing

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

Automated DNA sequencing

• Automated DNA sequencing makes use of the chain-termination technique
• A short length of DNA is chosen and inserted into a vector as a single strand of DNA
  • A primer is annealed to the start of the recombinant DNA

• During the incubation period, DNA polymerase is added to the recombinant DNA alongside a mixture of deoxyribose nucleotides (deoxynucleotides) containing all 4 bases
• Usually, DNA polymerase attaches to the primer and begins DNA replication of the single strand recombinant DNA
  • Hydrogen bonds form between the complementary bases on deoxyribose nucleotides

• However, a mixture of dideoxyribose nucleotides (dideoxynucleotides) are also present (containing all 4 bases)
• DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of DNA replication
• When there is a sufficient ratio of deoxynucleotides to dideoxynucleotides complementary DNA chains of varying lengths are produced
  • These chains can vary in length from one nucleotide to several hundred nucleotides

• 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

• Once the incubation period has ended and the dideoxynucleotides have bound to their complementary bases the DNA chains are removed from the template DNA
• The single-stranded DNA chains are then separated according to size using a specific type of electrophoresis that takes place inside a capillary tube
  • This type of electrophoresis technique 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
• An automated DNA sequencing machine can read roughly 100 different DNA sequences within 2 hours

The process of automated DNA sequencing. The sequence obtained is that of the developing DNA strand. It has a complementary sequence to the template strand that was inserted into the vector DNA.

Manual DNA sequencing

• Manual DNA sequencing follows a similar process to automated sequencing but there are some key differences:
  • A separate run is required for each type of dideoxynucleotide - ddNA, ddNT, ddNC and ddNG
  • The dideoxynucleotides are labelled using radioactivity instead of fluorescent dyes
  • After the incubation period, the four separate mixtures are added to separate wells in a gel and separated using gel electrophoresis
  • A Southern transfer is made using the electrophoresis gel and an autoradiograph is taken of the Southern transfer

Interpreting the results from manual DNA sequencing

• The DNA sequence can be interpreted using the autoradiograph
• Below each well there is a track of bands (DNA fragments) produced from DNA replication in the presence of each type of dideoxynucleotide (ddNA, ddNT, ddNC and ddNG)
• The band that moves the furthest distance is the smallest DNA fragment
• The smallest DNA fragment that can be formed from the chain termination technique is one nucleotide long so whichever track this band is present in determines the first base in the sequence of the developing strand
• The second smallest DNA fragment that has travelled the second furthest will determine the second base in the sequence and the third smallest DNA fragment that has travelled the third furthest will determine the third base in the sequence etc.

The base sequence of the test DNA can be read by identifying the distance each fragment has travelled, beginning with the fragment that has moved the furthest distance.