Genomic Informatics in the Healthcare System
Salvatore Volpe in Health Informatics, 2022
DNA is the code of all biological life on earth. Humans have sought to unravel its mysteries so that the origins of life itself may be revealed. The first sequencing methodology, known as Sanger sequencing, uses specifically manipulated nucleotides to read through a DNA template during DNA synthesis. This sequencing technology requires a specific primer to start the read at a specific location along the DNA template and record the different labels for each nucleotide within the sequence up to 1000–1200 base pairs (bps). Subsequently, an approach called shotgun sequencing was developed for longer read of sequences. In this approach, genomic DNA is enzymatically or mechanically broken down into smaller fragments and cloned into sequencing vectors in which cloned DNA fragments can be sequenced individually. The complete sequence of a long DNA fragment can be eventually generated by these methods by alignment and reassembly of sequence fragments based on partial sequence overlaps.
Precision medicine in ovarian carcinoma
Debmalya Barh in Precision Medicine in Cancers and Non-Communicable Diseases, 2018
Another method, known as the Sanger sequencing method, is centered on the principle that single-stranded DNA molecules that vary in length by just a single nucleotide can be separated from one another by using polyacrylamide gel electrophoresis. The stable laser beam excites the fluorescently labeled DNA bands and the light radiated is noticed by sensitive photodetectors. Automated DNA sequencing is freely automated by a variation of Sanger's sequencing method in which dideoxynucleosides castoff for each reaction is labeled with a differently colored fluorescent tag. Several commercial and noncommercial software packages can trim low-quality DNA traces automatically. DNA sequencing is still painstakingly the golden standard and the final experimental procedure for mutation detection. However, the costs for the initial investment and the difficulties for standardization and interpretation of ambiguous results have restricted its use to basic research laboratories. Because the chain-terminator tactics or Sanger approach is more efficient and this procedure involves fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it swiftly became the method of choice.
Evolution of the Molecular Microbiology Laboratory
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
The technology has a number of advantages in that it does not require the use of a gel, it is performed in a common microtiter plate, and is easy to perform. The disadvantage is that the length of sequence generated is small in comparison to sequences generated by Sanger sequencing. Approximately 30 bases constituted the longest sequence that we could generate with confidence, but there is some evidence that the length may be extended to 70 bases or more with modifications of the chemistry. This technology is particularly useful when variable regions that contain particular genetic information are identified. It is very useful for SNP analysis and may be used to identify single nucleotide polymorphisms associated with genetic diseases or resistance to antimicrobial agents.
STOX1 promotor region -922 T > C polymorphism is associated with Early-Onset preeclampsia
Published in Journal of Obstetrics and Gynaecology, 2022
Seyda Akin, Ergun Pinarbasi, Aslihan Esra Bildirici, Nilgun Cekin
DNA sequencing was conducted by Sanger sequencing method. A 3–5-μL aliquot of PCR product (collected directly or from re-amplification of excised SSCA bands) was used in a standard protocol for fluorescently labelled dideoxy-nucleotides (BigDye, Applied Biosystems, Life Technologies), with injection into a capillary electrophoresis instrument (ABI 3500, Life Technologies) for separation and detection. The sequences obtained were compared with the reference sequence NC 000017 (www.ncbi.nlm.nih.gov), and deviations were recorded as mutations or polymorphisms. Chromas Lite 2.6.6 software was used to display the results and to scan the changes in the array. Also, DNA sequencing was employed using the next generation sequencing system Illumina technology. IGV 2.6.3 (Archived) program was utilised to display the results and to observe the changes in the array.
Effects of CYP3A4*1G and CYP3A5*3 polymorphisms on pharmacokinetics of tylerdipine hydrochloride in healthy Chinese subjects
Published in Xenobiotica, 2019
Sufeng Zhou, Mingxue Tao, Yuanyuan Wang, Lu Wang, Lijun Xie, Juan Chen, Yuqing Zhao, Yun Liu, Hongwen Zhang, Ning Ou, Guangji Wang, Feng Shao, Jiye Aa
DNA from K2EDTA-treated venous blood samples was extracted using a TIANGEN DNA Blood kit (Tiangen Biotech Co., Ltd, Beijing, China). Polymerase chain reaction (PCR) was used to identify the genotype of CYP3A4*1G and CYP3A5*3. The sequences of PCR primers were designed by the company of Synbio Technologies (Suzhou, China). The forward (F) and reverse (R) primers were as follows: F1 (5′-CAGAG CCAGCACGTTTTACA-3′) and R1 (5′-CTCCTTTCAGCTCTGTCCGA-3′) for CYP3A4*1G; F2 (5′-GCACTTGATGATTTACCTGCCT-3′) and R2 (5′-TTCACTAGCCCGATTCTGCA-3′) for CYP3A5*3. The PCR was performed in a reaction mixture containing 12.5 μL of Phantaturbo master mix (VazymeBiotech Co., Ltd, Nanjing, China), 1 μL of each primer, 50 ng genomic DNA and ddH2O in a total volume of 25 μL. Amplification conditions were an initial denaturation at 96 °C for 5 min, 35 cycles of denaturation for 30 s at 96 °C, annealing at 58 °C for 30 s and extension for 30 s at 72 °C, with a final extension for 7 min at 72 °C. Then, the genotyping was performed by Sanger sequencing. Sanger sequencing is a technique for DNA sequencing based upon the random chain-termination method which requires the addition of ddNTPs to form four separate DNA strands ended with A, T, C and G with various sizes. Then, the resulting DNA fragments are analyzed by electrophoresis using a denaturing polyacrylamide-urea gel and the DNA bands are visualized by autoradiography. The Sanger sequencing results could indicate whether the peak of detected site is heterogeneous or homogenous and then the alteration of the detected site would be examined so the genotype is figured out.
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].
Related Knowledge Centers
- Dideoxynucleotide
- DNA Polymerase
- DNA Replication
- DNA Sequencing
- Genome
- In Vitro
- Nucleoside Triphosphate
- Nucleotide
- Primer
- Hydroxy Group