Sanger sequencing (and other PCR-based approaches) DirectDetectionofDNAMutationsbyare starting to give way to next-generation sequencing (discussed later), particularly when the analysis of large genes or multiple genes is required. Nonetheless, even PCR analysis, which involves exponential amplification of in 2016, Sanger sequencing remains the “gold standard” DNA, is now widely used in molecular diagnosis. If RNA for many genotyping assays. is used as the substrate, it is first reverse-transcribed to • Another approach for identifying mutations at a specific obtain cDNA and then amplified by PCR. This method nucleotide position (say, a codon 12 mutation in the involving reverse transcription (RT) often is abbreviated as KRAS oncogene that converts glycine [GGT] to aspartic RT-PCR. To amplify a DNA segment of interest, two acid [GAT]) would be to add fluorescently labeled primers that bind to the 3′ and 5′ ends of the normal nucleotides C and T to the PCR mixture, which are sequence are designed. By using appropriate DNA poly-complementary to either the wild-type (G) or mutant merases and thermal cycling, the target DNA is greatly (A) sequence, respectively. Because these two nucleoamplified, producing millions of copies of the DNA tides are labeled with different fluorophores, the fluosequence between the two primer sites. The DNA sequence rescence emitted by the resulting PCR product can be of of the PCR product can then be analyzed in several ways: one or another color, depending on whether a C or a T • Sanger sequencing, named after its inventor, Nobel-becomes incorporated in the process of primer extenlaureate Frederick Sanger, has for many years been the sion (
Fig. 7.40 ). The advantage of this allele-specific “workhorse” of genome sequencing, including the orig-extension strategy is that it is more sensitive than Sanger inal Human Genome Project. Here, the amplified DNA sequencing, allowing for the identification mutated is mixed with a DNA polymerase, a DNA primer, nucle-DNA sequences even if they make up a small fraction otides, and four dead-end (di-deoxy terminator) nucleo-(~1%) of the total DNA (as may be the case in clinical tides (A, T, G, and C) labeled with different fluorescent specimens obtained from patients suspected of harbor-tags. The ensuing reaction produces a series of DNA ing a malignancy). An ultrasensitive variant of this molecules of all possible lengths up to a kilobase or so, assay, known as droplet digital PCR, literally separates each labeled with a tag that corresponds to the base at the template DNA (or cDNA) into thousands to millions which the reaction stopped because of the incorporation of microscopic oil “droplets,” such that any given http://ebooksmedicine.net
Fig. 7.40 Allele-specificpolymerasechainreaction(PCR)analysisformutationdetectioninaheterogeneoussamplecontaininganadmixtureofnormalandmutantDNA.Nucleotidescomplementarytothemutantandwild-typenucleotidesatthequeriedbasepositionarelabeledwithdifferentfluorophoressuchthatincorporationintotheresultingPCRproductyieldsfluorescentsignalsofvariableintensityinaccordancewiththeratioofmutanttowild-typeDNApresent.
droplet contains no more than one strand of template DNA, along with requisite PCR reagents. Each such “minireactor” then generates its own PCR product, which can be digitally integrated and analyzed for the presence of mutant alleles. By assessing each template individually in parallel, rather than in a single PCR reaction, droplet digital PCR has exquisite sensitivity, being able to detect as little as 1 mutant DNA strand in 10,000 or more wild-type molecules.