DNA Sequencing

Determine the order of nucleotides

A DNA molecule is made up of two sugar phosphate spines forming a double helix. The spines are connected by base pairs of nucleotides. Adenine (A) always pairs with thymine (T) and cytosine (C) always pairs with guanine (G).

DNA sequencing determines the order of deoxyribonucleotides, or simply “nucleotides” or “bases”, within a DNA molecule. The sequence of nucleotides along the length of a DNA chain contains the instructions for replicating, building, and maintaining the cells of an organism.

By comparing ordinary versions of genes with disease-causing versions, DNA sequencing reveals valuable information about the role of inheritance and susceptibility to disease. It is also employed in medical diagnosis and treatment through gene therapy.

Chain termination sequencing

“Chain termination sequencing” or “Sanger sequencing” is a DNA sequencing technique based on methods that utilised radioactive substances. More recently, a faster and safer version of Sanger sequencing that uses fluorescence instead of radioactivity is routinely employed in genetic studies. The process can be arranged into four steps.

1) The sample is amplified by mixing a DNA sample with a solution containing nucleotides, DNA polymerases, and primers. The polymerases catalyse many new copies of the DNA molecule in a replication process called polymerase chain reaction (PCR). Primers are short strands of DNA that initiate DNA chains and serve as a consistent starting point for new copies.

An automated Sanger sequencer utilises gel electrophoresis, an excitation source, a photodetector, and a computer system in order to depict the DNA sequence of a specimen.

2) By adding a type of nucleotide known as ddNTP to the solution, PCR is terminated. ddNTPs are near-identical versions of ordinary nucleotides, denoted as ddCTP, ddGTP, ddATP, and ddTTP. Each of the four ddNTPs are tagged with a different fluorophore so that when they are illuminated by a given wavelength, they fluoresce at a characteristic colour. A growing DNA chain can randomly bond with a ddNTP instead of a regular nucleotide. This terminates the chain and results in a partial copy of DNA, or “DNA fragment”. Many such fragments are terminated at random points and therefore have a range of physical lengths that end with a fluorescent ddNTP.

3) The next step is to arrange the fragments in order of size using a method called capillary gel electrophoresis. The fragments travel through the pores of a gel at speeds that are inversely related to their lengths. The DNA fragments are negatively charged. As a result, they can be attracted toward the positive electrode of a capillary containing the porous gel in order of increasing size. 

4) The fragments individually pass through laser illumination and the resulting fluorescent signals of the ddNTPs at the end of each fragment are detected. The DNA sequence is read by the computer from bottom to top, i.e. the smallest fragments to the longest.

An example of a DNA sequence is

Example DNA Sequence

Each three-letter sequence, dubbed a “codon”, specifies which amino acid should be added next to a growing chain of amino acids to form a protein. This continues until a STOP codon is reached, signalling that the protein is complete. Occasionally, DNA mutations add a STOP codon in the wrong place, resulting in a useless protein. Such proteins cause around 10% of all genetic diseases.

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