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    Rights: University of Waikato
    Published 29 June 2018 Referencing Hub media

    DNA sequencing is the process used to determine the exact sequence of the nucleotides in a strand of DNA. DNA sequencing is used to determine the nucleotide sequence of specific genes, larger genetic regions, whole chromosomes or the entire genome of an organism.



    DNA is found in almost every cell of every living organism.

    DNA sequencing is the process used to determine the order of the bases in a strand of DNA. The order of bases varies between organisms and codes for their unique characteristics.

    In order to sequence DNA, it must first be broken into smaller pieces and amplified to create multiple copies.

    PCR is usually used to do this.

    The DNA is then heated to above 90°. This denatures the DNA, which means the hydrogen bonds between the base pairs break and the strands separate. One of these strands becomes the template for DNA sequencing.

    The thousands of copies of single-stranded DNA created by PCR are then mixed with a solution containing DNA primers, free DNA nucleotides A, T, C and G, terminator nucleotides and the enzyme DNA polymerase.

    The terminator nucleotides are specially modified DNA nucleotides that have been chemically altered to become dideoxynucleotides and are labelled with coloured fluorescent markers.

    The solution also contains a buffer to ensure the optimum pH for the reaction.

    The temperature is then lowered, allowing the primers to attach to the DNA fragments. This process is called annealing. Primers are pieces of single-stranded DNA specifically designed to match part of the DNA to be sequenced. They are required to start the process of making new DNA.

    The temperature is increased again, and the DNA polymerase enzyme binds to the primer. It then starts making a new strand of DNA by adding nucleotides in the order determined by the bases on the template strand, with A matching T and C matching G etc.

    The process continues until one of the terminator nucleotides is randomly added instead of a normal nucleotide. Once this happens, the DNA polymerase cannot continue making new DNA. The process stops, and the DNA polymerase falls away.

    As there are multiple copies of the DNA template strand, many new fragments of DNA are made.

    The insertion of the terminator nucleotides occurs in a random fashion, so many DNA fragments of different lengths are synthesised.

    The mixture is heated again to denature the DNA, so the newly made fragments are separated from the template strands.

    The process produces multiple fragments of DNA of varying lengths ending at each base position of the original template. The length of each fragment depends on when the terminator nucleotide was added.

    To read the sequence of the bases in the original DNA, the DNA fragments are separated by length by a process called electrophoresis.

    The DNA mixture is added to capillary tubing containing an electrophoresis gel, and an electric current is applied. The current draws the fragments along the tubing according to their size. The shorter fragments move through the gel more easily so reach the end more quickly than the longer fragments.

    Note that this representation has been simplified – in reality, hundreds of copies of every length fragment would be travelling together.

    As the fragments reach the end of the capillary tube, an optical scanner picks up the fluorescent marker on the terminator nucleotides. Each of the four types of terminator nucleotides is labelled with a different colour, representing each of the four DNA bases. The shortest DNA fragments will be scanned first.

    The scanner records the sequence of colours that fluoresce as the fragments come past. By reading the order of the colours, the sequencing machine can determine the order of the bases in the original DNA by converting the colours into the letters A, C, T and G.