Sequencing-The principle

Probably the most important technique available to the molecular biologist is DNA sequencing, by which the precise order of nucleotides in a segment of DNA can be determined. Two different techniques for sequencing were developed almost simultaneously-the chain termination method by F. Sanger and A.R. Coulson in the Uk and the chemical degradation method by A. Maxam, and W. Gilbert in the USA.

The Sanger-Coulson Method

The ‘Sanger’, ‘dideoxy’, or ‘chain-termination’ method of DNA sequencing relies on the enzymatic synthesis of DNA in vitro in the presence of chain-terminating inhibitors.

(a) The primer

The first step in a chain termination sequencing experiment is to anneal a short oligonucleotide primer on to the recombinant molecule (Plasmid containing the DNA to be sequenced). This primer will act as a starting point for complimentary strand synthesis reaction carried out by the Klenow fragment of DNA polymerase. The primer anneals to the vector at a position adjacent to the polylinker.

(b) Synthesis of the complementary strand

The chain synthesis is started by adding the enzyme plus each of the four deoxynucleotides (dATP, dCTP, dGTP, dTTP). In addition a single modified nucleotide is also included in the reaction mixture. This is a dideoxynucleotide (e.g. dideoxy dCTP) which can be incorporated into the growing polynucleotide strand just as efficiently as the normal nucleotide, but which blocks further strand synthesis. This is because the dideoxy-nucleotide lacks the hydroxyl group at the 3’ position of the sugar component. This group is needed for the next nucleotide to be attached; chain termination therefore occurs whenever a dideoxynucleotide is incorporated by the enzyme.

 

(c) Four separate reactions result in four families of terminated strands

The strand synthesis reactions is carried out four times in parallel (ddATP, ddCTP, ddGTP and ddTTP). The result will be four different distinct families of newly synthesized polynucleotide, one family containing strands all ending in dideoxy ATP, one of strands ending in dideoxy TTP, etc. The next step is to separate the components of each family so the lengths of each strand can be determined. This can be achieved by very thin polyacrylamide gel electrophoresis. The gel contains urea, which denatures the DNA so the newly synthesized strands dissociate from the templates. In addition the electrophoresis is carried out at a high voltage, so the gel heats up to 600C and above, making sure the strands do not reassoiciate in any way.

Each band in the gel will contain only a small amount of DNA, so autoradiography has to be used to visualise the results. The label is introduced into the new strands by including a radioactive deoxynucleotide (32P- or 35S-dATP) in the reaction mixture for the strand synthesis step earlier in the experiment.

 

Advances in DNA sequencing

DNA sequencing by radioactive labelling is thankfully no longer necessary due to the advent of dye-terminator chemistry, whereby a single strand of DNA can be labelled with a fluorescent dye corresponding to the base at the 3'end of the fragment.

The fluorescent sequencing reaction is similar to a PCR reaction in that template DNA is copied to produce new strands, commencing at the site of an annealed primer. In a dye terminator sequencing reaction however, only one primer is used and, as well as the usual deoxynucleotide triphosphates (dNTPs) there are four dye-labelled dideoxynucleotides (ddNTPs). When a ddNTP is incorporated into the growing DNA strand, synthesis is terminated due to the absence of a hydroxyl group. The final products of the sequencing reaction consist of a set of fragments of different lengths, fluorescently labelled at the 3’end. These products can be electrophoresed on a polyacrylamide gel, which is able to separate fragments, which differ, by only one base pair.

Each of the four ddNTPs has a different fluorescent dye attached which emits light at a different wavelength when excited by a laser. The resulting fluorescence can be detected by a CCD camera and translated into a chromatogram. As the fluorescently labelled extension products from a sequencing reaction are electrophoresed past the laser detection area of a gel, each discrete base can be "called". Over the course of a seven-hour run, upto 600 bases can be accurately called, depending on the quality of the template.

 

 

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