Molecular Genetics and Diagnostic Testing
Merlin G. Butler, F. John Meaney in Genetics of Developmental Disabilities, 2019
Determining the order of the nucleotide bases in DNA is typically done by the dideoxy method (chain-termination of Sanger method) (10). DNA polymerase proceeds to synthesize DNA strands of various lengths, stopping DNA replication at one of four bases and determining the resulting fragment lengths. In addition to the DNA template and the enzyme, sequencing reactions contain a primer to bind to the DNA, four deoxynucleotides to be incorporated into the new DNA strand, one labeled deoxynucleotide (using radioactivity or fluorescent dye), and one dideoxynucleotide that terminates the strand wherever it is incorporated. The concentration of the nucleotides is adjusted, so that dideoxynucleotides are incorporated into each position in which that nucleotide occurs producing a collection of DNA fragments, each with a dideoxynucelotide at the end. The fragments are separated by electrophoresis and the positions of the nucleotides visualized by autoradiography or by an automated laser-equipped instrument to determine the sequence (Fig. 7).
Cancer Informatics
Trevor F. Cox in Medical Statistics for Cancer Studies, 2022
In Sanger sequencing, four inhibitors of chain elongation in the process of DNA polymerisation are used. They are dideoxynucleotides (modified nucleotides), denoted by ddATP, ddCTP, ddGTP, ddTTP, and they stop a chain from further elongation at an A nucleotide (denoted dATP), a G nucleotide (dGTP), a C nucleotide (dCTP) and a T nucleotide (dTTP) respectively. Polymerase chain reaction is carried out with four separate samples of the DNA as described above, but with one of dideoxynucleotides added to each sample. So the first sample might have ddCTP added but in a smaller concentration than the “C” nucleotide, and similarly for the other three samples. During PCR for the strand (or fragrant) building caused by the polymerase in the first sample, each fragment will build as normal with the dATPs, dCTPs, dGTPs, dTTPs being added until, at random, a ddCTP is added and so the fragment will terminate at that point, and similarly for the other three samples. After the PCR is finished, each of the four samples will contain fragments of various lengths, all terminating in their added dideoxynucleotide.
Preimplantation Genetic Diagnosis for Single Gene Disorders
Carlos Simón, Carmen Rubio in Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Genotyping of the amplified products can be performed by means of different strategies, with minisequencing the most frequently used method for the detection of point mutations (23). In the minisequencing technique, a primer extension reaction is performed, allowing rapid and accurate detection of point mutations. The minisequencing primer is designed to anneal one base before the target site, and it can only be elongated with one specific dideoxynucleotide. The four different dideoxynucleotides are labeled with different fluorochromes, and the products can be analyzed on an automated DNA sequencing system. Other strategies such as amplification refractory mutation system (24), restriction enzyme digestion (25), and real-time PCR (26) have been also applied in PGT-M. Small deletions and duplications can also be detected by sizing of PCR products from specific regions containing the mutation under study.
Common mutations of interest in the diagnosis of amyotrophic lateral sclerosis: how common are common mutations in ALS genes?
Published in Expert Review of Molecular Diagnostics, 2020
Benedetta Perrone, Francesca Luisa Conforti
Genetic testing for SOD1, TARDBP, and FUS genes includes first and second-generation DNA sequencing methods. Sanger sequencing is the ‘first-generation’ DNA sequencing method, widely used in ALS diagnosis, first emerged in 1977 [110]. Sanger Sequencing is known as the chain termination or the dideoxynucleotide or the sequencing by synthesis method. It consists of using one strand of the double-stranded DNA as a template to be sequenced. This sequencing is made using chemically modified nucleotides called dideoxynucleotides (dNTPs). These dNTPs marked for each DNA bases by ddG, ddA, ddT, and ddC also include a fluorescent marker (A is indicated by green fluorescence, T by red, G by black, and C by blue). The fluorescent dideoxynucleotides (dNTPs) are used for elongation of nucleotide, once incorporated into the DNA strand they prevent the further elongation. Then, we obtain DNA fragments ended by a dNTP with different sizes and fragments, separated according to their size by capillary electrophoresis. A laser within the automated machine used to read the sequence detects a fluorescent intensity that is translated into a ‘peak’ revealing heterozygous or homozygous variants within a sequence [111].
Next-generation sequencing for the diagnosis of hepatitis B: current status and future prospects
Published in Expert Review of Molecular Diagnostics, 2021
Selene Garcia-Garcia, Maria Francesca Cortese, Francisco Rodríguez-Algarra, David Tabernero, Ariadna Rando-Segura, Josep Quer, Maria Buti, Francisco Rodríguez-Frías
First-generation Sanger sequencing uses specific chain-terminating nucleotides (dideoxynucleotides, ddNTPs) labeled with different fluorochromes that lack the 3ʹ-OH group, which results in the termination of the growing DNA chain at that position [41]. This produces a mixture of DNA fragments of different sizes with the four fluorochrome labels that can be detected when the fragments are separated by size in capillary electrophoresis-based sequencers [4].
Practical applications of DNA genotyping in diagnostic pathology
Published in Expert Review of Molecular Diagnostics, 2019
Another useful method that can be used for identification of tissue is the single nucleotide polymorphism (SNP) Array. The principle behind this technique is the identification of differential patterns of SNPs across various regions of the genome, including highly polymorphic regions such as HLA loci, or other less variable regions. As mentioned previously, the overall statistical likelihood of an SNP between two individuals is 1 in every 1000 nucleotides. Thus, by interrogating a sufficiently broad array of sequences, an adequate amount of differential SNPs between two individuals can be achieved to allow determination of identity with sufficient confidence [28–30]. Detection is usually accomplished either by DNA microarray chip or by fluorescent detection of differential base pair incorporations. The SNaPshot SNP Minisequencing System (ThermoFisher) is one of the more widely used SNP array products, and allows for multiplexed interrogation of numerous SNPs. The process begins with PCR amplification of the region of interest, followed by hybridization with a primer that is complementary to the sequence immediately upstream of the variable nucleotide in question. Following annealing, the primer is extended by a single fluorescently labeled dideoxynucleotide, with differential color fluorescence for each nucleotide. Subsequent capillary electrophoresis with laser detection allows for the identification of the variable nucleotide in the SNP on the basis of fluorescence color (Figure 1(b)). When multiplexed, this can be used to simultaneously identify tens of thousands of SNPs from individual amplicons, and has been used in forensic identification cases [31–33], while other lab-developed SNP arrays have the ability to analyze over 100 SNPs in a single combined assay [34]. It is worth noting that next-generation sequencing technology has remarkably advanced our sequencing capability in providing ultra-throughput of SNP data for genotyping (genotyping-by-sequencing or GBS). GBS has been demonstrated to be a robust and cost-effective method, capable of producing thousands to millions of SNPs across a wide range of species, although currently being used primarily in plant/crop breeding fields [35,36].
Related Knowledge Centers
- DNA Polymerase
- DNA Sequencing
- Phosphodiester Bond
- Polymerase Chain Reaction
- Pyrophosphate
- Recombinant DNA
- Sanger Sequencing
- Hydroxy Group
- Maxam–Gilbert Sequencing
- Electrophoresis