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An Introduction to the Immune System and Vaccines
Published in Patricia G. Melloy, Viruses and Society, 2023
The development of PCR has revolutionized many aspects of science involving detection of small amounts of any specific DNA or RNA, not just viral DNA or RNA. Describe three major applications of PCR besides viral testing. You can use the interactive website on PCR applications created by the LabXChange group affiliated with Harvard University: www.labxchange.org/library/items/lb:LabXchange:bc4846e2:lx_simulation:1
Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
To amplify DNA using PCR, a segment of DNA is placed in solution containing primers (short sequence of single stranded DNA), free DNA nucleotides, DNA polymerase (an enzyme that catalyses the synthesis of DNA molecules) and other ingredients. Figure 10.1 illustrates the process where a target section of DNA is to be amplified. The solution is heated to over 90∘C to separate the DNA strands. The temperature is then lowered and primers attach themselves to the ends of the target section of DNA. The polymerase builds two new strands starting at the primers, but note only one end is extended. Heating again separates the strands into four separate ones. On lowering the temperature, primers again attach themselves and the polymerase extends the strands. There are now four strands of DNA. The next step will produce eight strands of the target DNA. The process continuously repeats, producing strands and so easily making many copies of the target DNA, which can then be used in experimentation.
Genetics and metabolic disorders
Published in Jagdish M. Gupta, John Beveridge, MCQs in Paediatrics, 2020
Jagdish M. Gupta, John Beveridge
PCR allows amplification of a specific DNA sequence which can then be further analysed. It can be used to analyse small or impure samples. Genetic probes are nucleic acid bases arranged in a sequence which allow detection of whether genomic sequence is present, deleted or altered. In Southern blotting the DNA sample is cut into fragments at specific sites by enzymes. The fragments are size sorted by electrophoresis and analysed by radiolabelled gene probes. In situ hybridization is used to detect deletions and rearrangements of chromosomal DNA. Chromosomal analysis is useful for gross abnormalities including large deletions or duplications/insertions of chromosomal material.
Diabetic retinopathy progression associated with haplotypes of two VEGFA SNPs rs2010963 and rs699947
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Haider Ali Alnaji, Rabab Omran, Aizhar H. Hasan, Mohammed Qasim Al Nuwaini
Genotyping. Genomic DNA was extracted from whole blood in EDTA tubes using G-spin™ Total DNA Extraction Mini Kit (iNtRON Biotechnology, Korea) and stored at −20°C in the biotechnology laboratory of Babylon University until the genotyping. The genotyping of the SNPs in the study were tetra primers ARMS-PCR technique. In brief, the outer primers of rs2010963 and rs699947 SNPs generate the confirmative amplicon for the 5’ UTR and promoter region where the SNPs resides. The inner reverse primers were designed for the mutant allele by mismatching the 3’ end of the primer. Also, the third nucleotide of the 3’ end was mismatched to increase the specificity. The PCR steps were as follows: 40 cycles of denaturation at 95°C for 35 s and annealing at 65°C decreased gradually every cycle to reach the lowest annealing at 58°C for 40 s and 72°C for extension and final extension for 40 s and 7 min respectively. The PCR cycles are preceded by initial denaturation at 95°C for 2 min for activation. Followed by agarose gel electrophoresis (2% and 75 V) for 1 hour. The SNPgen® tool was used to design rs2010963 primers, whereas rs699947 primers were obtained from an Elfaki et al. study [16], as shown in Table 1.
The new normal: a UK fertility clinic experience of universal RT-PCR SARS-CoV-2 testing
Published in Human Fertility, 2023
Ektoras X. Georgiou, Victoria Ryder, Julia Paget, Richard Banks, Ying C. Cheong
The Complete Fertility Centre (CFC), located in the city of Southampton, ceased starting fresh cycles on 3rd April 2020 and re-opened on 12th May 2020. In addition to the routine use of personal protective equipment and social distancing measures, a testing plan was designed to screen both elective and non-elective patients in light of emerging evidence of the broad spectrum of disease presentation (Kronbichler et al., 2020). A clinic level decision was taken for universal testing by real time polymerase chain reaction (RT-PCR), with evidence supporting this as the gold-standard test for diagnosis (Böger et al., 2021). RT-PCR involves the use of a reverse transcriptase enzyme to convert ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), with amplification detected by fluorescence. The process uses a primer allowing amplification of very specific nucleic acid sequences, in this case that of SARS-CoV-2, hence its high degree of accuracy. At the time of this decision, lateral flow testing was not available.
Critical insight into recombinase polymerase amplification technology
Published in Expert Review of Molecular Diagnostics, 2022
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.