Laboratory techniques to study the cellular and molecular processes of disorders
Louis-Philippe Boulet in Applied Respiratory Pathophysiology, 2017
The advance of PCR with high throughput platforms played a tremendous role in realizing the Human Genome Project that documented the entire human genome sequence [24]. The project led to the identification and documentation of genetic variations. These variations in turn become landmarks on the map of the human genome and allow genetic studies to be carried out. Genotyping, the determination of the alleles present in an individual, becomes the aim of many genetic studies. In asthma research, genotyping enables linkage studies to detect chromosomal regions housing asthma or asthma-trait–related genes (e.g., 5q31–33, 6p21, 12q13–q24, and 17q12–q21) [25] and genetic association studies (both genome-wide or candidate genes) to identify asthma or as, thma-trait–related genes (e.g., ADAM33, ADRB2, CD14, GSTP1, HLA-DRB1, HLA-DQB1, IL4, IL4R, IL10, IL13, LTA, MS4A2, STAT6, and TNFA) (PMID:18301422). It can be insightful to group disease-associated genes into classes according to biological functions. For example, the grouping of 65 asthma-associated genes revealed that more than half of the genes (54%) have an immune response function and 18% of the genes are involved in tissue remodeling. Finally, 17 genes (28%) fall into the “other” category suggesting that diverse biological pathways are also involved in asthma pathogenesis (Figure 3.7) [26]. More research is needed to identify and understand how these biological pathways underlie asthma pathogenesis.
Hypertrophic Cardiomyopathy
Andreas P. Kalogeropoulos, Hal A. Skopicki, Javed Butler in Heart Failure, 2023
Cascade screening of relatives of patients with HCM is essential. First-degree relatives of the proband should undergo serial intermittent EKG and echocardiography in the absence of a known disease-causing genetic mutation.30 Phenotypic screening frequency depends on the age of the relative, with children aged 7–18 years old requiring screening every 12–18 months, with relatives >18 years old tested only every 3–5 years. Genetic analysis is useful to confirm the genetic etiology and to differentiate isolated HCM from other syndromic conditions associated with it (such as Danon disease, Fabry disease, amyloidosis, and mitochondrial cardiomyopathy). A significant portion of HCM can also be attributed to de novo variants, compound variants, or pathogenic variants in multiple genes; each of these scenarios conveys a different risk to a proband's relatives.40 Whereas genotyping is a useful tool for familial risk stratification, there are serious pitfalls and implications, especially on young individuals, that mandate thorough professional genetic counseling and understanding of the limitations of the technique.
Identification of Genes Underlying Polygenic Obesity in Animal Models
Claude Bouchard in The Genetics of Obesity, 2020
Genotyping means to identify which of the two parental strains has contributed alleles to a specific locus in each F2 or backcross animal (Figure 2). The two most common methods for genotyping are polymerase chain reaction (PCR) assay of simple sequence repeats (SSRs) and RFLPs of complementary DNA (cDNA) or genomic clones. The most common SSR consists of a variable number of (CA)n repeats, is present at approximately 100,000 copies per genome, and is so polymorphic that any two inbred mouse or rat strains will, on average, be polymorphic at 50% of existing SSR loci.23,28 Genotyping with SSR primers is faster and cheaper than with RFLPs, in part because reactions can be performed in 96-well PCR plates and in part because more than 1500 mouse and 200 rat SSR primer pairs are available at low cost from Research Genetics (Birmingham, AL). Genotyping by RFLPs requires that one acquire and prepare cDNA or genomic clones, prepare Southern blots, and probe the blots. Genotyping by RFLPs remains useful when one needs to test a specific candidate gene for linkage to a trait.
High dose methotrexate in adult Egyptian patients with hematological malignancies: impact of ABCB1 3435C > T rs1045642 and MTHFR 677C > T rs1801133 polymorphisms on toxicities and delayed elimination
Published in Journal of Chemotherapy, 2022
Abdel-Hameed I. M. Ebid, Ahmed Hossam, Mosaad M. El Gammal, Sameh Soror, Nadia O.M Mangoud, Mohamed Adel Mahmoud
The extraction of DNA was done using GeneJet Whole Blood Genomic DNA purification Mini Kit (Thermo Fisher Scientific, USA). The DNA samples were stored at −80 0C until genotyping. Assessment for purity and concentration of the extracted DNA samples were done using ‘Quawell Q5000 nanodrop’ (Quawell Technology, Inc., San Jose, CA). DNA purity of A260/280 ranged from 1.7 to 1.9 was considered pure for genotyping. The genotyping was done by the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA) for our selected SNP assays: rs1801133 (assay ID = C___1202883_20, assay type = functionally tested) and rs1045642 (assay ID = C___7586657_20, assay type = drug-metabolizing enzyme) together with TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA) on the Rotor-Gene Q real-time thermocycler instrument (Qiagen, Hilden, Germany). The reaction mixture composed of 10 μL of master mix, one μL of DNA sample, 0.5 μL SNP assay, and finally DNase Free water to a total volume of 20 μL. Some samples were selected randomly to assess the accuracy of the genotyping process. Rates of concordance were 100% for the tested samples.
Association between AKT2 gene polymorphism and polycystic ovary syndrome: a case-control study
Published in Gynecological Endocrinology, 2021
Jie Li, Sien Mo, Yan Sun, Hua Huang, Shujia Wang
The whole blood was collected from all participants after acquiring informed consent. Genomic DNA was extracted from peripheral blood leukocytes using a DNA isolation kit following the manufacturer’s instructions (Sangon Biotech, Shanghai, China). The genotyping was mainly based on the SNPscan method (Genesky Biotechnologies Inc., Suzhou, China). The details were presented in other studies (http://biotech.geneskies.com/en/index.php/Index/fuwuer/id/29) [17–18]. Briefly, this method was a multi-gene mutation screening technology, which ameliorated the previous multiplex ligation-dependent probe amplification technology to some extent. During the process of this method, four different fluorescent dyes were applied. In addition, the ligations were lengthened, which could help to detect more SNPs simultaneously. In order to ensure the veracity of the genotyping, quality control assessment was also performed during the genotyping process. The laboratory personnel were blinded to the case-control status of the samples, with a blank control used for all PCR amplifications. Finally, 10% of samples from patients and controls were randomly selected to evaluate the quality of genotyping, which showed 100% concordance.
Critical insight into recombinase polymerase amplification technology
Published in Expert Review of Molecular Diagnostics, 2022
Mustafa Ahmad Munawar
Kary Mullis developed the revolutionary technique of polymerase chain reaction (PCR) in 1983 [1], and its modern version has evolved through several of the improvements in the chemistry and machinery. PCR is a cyclic process of amplifying template DNA through primers, polymerase, deoxynucleoside triphosphates (dNTPs) (nucleotides or building blocks), magnesium ion, buffer, and two to three different incubation temperatures. PCR utilizes (1) a high temperature such as 95°C to convert double-stranded DNA into single-stranded DNA (melting), then (2) a temperature around 55°C to facilitate annealing of a primer pair with target DNA, and (3) a temperature around 70°C to extend annealed primers through polymerase. A typical PCR comprises 30–40 cycles, and it exponentially amplifies target DNA into millions of amplicon copies. PCR is also utilized to amplify RNA. RNA template is first converted into complementary (c)DNA through reverse transcriptase (RT) and then amplified through PCR. In summary, PCR is utilized for qualification, absolute quantitation, and relative quantification of target nucleic acid, and it is used massively in many fields including diagnostics, forensics, and agriculture. PCR applications include detection of pathogens or contaminants, genotyping, relative quantification of gene expression, molecular cloning, sequencing, methylation detection, and site-directed mutagenesis.
Related Knowledge Centers
- Allele
- Amplified Fragment Length Polymorphism
- Bioassay
- DNA
- DNA Sequencing
- Polymerase Chain Reaction
- Restriction Fragment Length Polymorphism
- Genotype
- Random Amplification of Polymorphic DNA
- Allele-Specific Oligonucleotide