There are times when PCR is not sufficient to provide the answers that we need about a patient’s condition. For example, tumor suppressor genes can be deactivated through numerous point mutations; therefore full gene sequencing is needed. Sanger sequencing is still the most widely used sequencing assay.
Sanger sequencing uses modified nucleotides (dideoxynucleotides or ddNTP) in a PCR reaction. The bases used in Sanger sequencing are fluorescently labeled and modified such that when the base is incorporated into the DNA strand, synthesis of that strand stops. The ddNTPs are present in the reaction in low concentrations alongside higher concentrations of normal bases. This allows thousands of DNA strands to be made, ending at a different point when a modified base happens to be incorporated into their strand. The sequence of the strands is then determined using a capillary electrophoresis instrument. The sequence can then be compared to a known sequence to determine if there are mutations in the DNA analyzed.
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