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Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Matthew Lanktree, Amirreza Haghighi, Xueweng Song, York Pei
The choice of DNA polymerase and PCR conditions is critical for successful PCR amplification as some enzyme mixes are more robust than others. Although PKD1gene-specific LR-PCR primers have been carefully designed to differentiate the true PKD1 gene from its six pseudogenes, it remains difficult to efficiently amplify all fragments under standard PCR conditions because of the high GC content and sequence complexity.20,22 KOD Xtreme Hot Start DNA Polymerase (EMD Millipore) is an optimized PCR enzyme for the amplification of long (up to 24 kb), GC-rich (up to 90% GC content) and crude (minimally processed) DNA templates. By using KOD Xtreme, we could successfully amplify the extreme GC richness of PKD1 exon 1 fragment (2278 bp), and the longest PKD1 fragments (exon 2–15 (11,486 bp)) with minimal optimizations, which routinely failed when other commercial LR-PCR polymerases for GC-rich templates were used. The detailed PCR amplification conditions for the seven LR-PCR fragments are described in Table 14.3. Using the LR-PCR products as the input DNA, primers directed to flank each exon can be used for capillary sequencing with input DNA free from pseudogene sequence. Alternatively, the LR-PCR products can be advanced for high-throughput sequencing.
Genetic and genomic investigations
Published in Angus Clarke, Alex Murray, Julian Sampson, Harper's Practical Genetic Counselling, 2019
Genetic laboratories have used several methods to determine the DNA sequence of specific genes. Most have depended upon the PCR to amplify the specific fragment of DNA that is of interest, from among the rest of the patient's DNA obtained from their blood or other tissues. DNA primers are constructed to flank the stretches of sequence in which mutations are often found and then these are amplified using PCR to generate a sufficient quantity of ‘pure’ (or at least ‘clean’) material for analysis. Amplification of DNA by cloning can also be used. The DNA from one or a few PCRs will then be sequenced by the conventional (Sanger) method. The amplified DNA is copied by a DNA polymerase in four separate reactions with a small proportion of dideoxynucleotides (ddNTPs) present in the standard mix of nucleotides, and with a fluorescent label attached to one of the four ddNTPs. When a ddNTP is incorporated, that molecule can no longer be extended and so the length of the molecule with the ddNTP at its end identifies the nucleotide at that specific position in the gene.
The Single-Stranded DNA Binding Protein of Bacteriophage T4
Published in James F. Kane, Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
Daniel H. Doherty, Peter Gauss, Larry Gold
In summary, total DNA synthesis using nicked double-stranded DNA molecules as templates requires the seven protein system. These replication proteins polymerize nucleotides into DNA using a DNA or RNA primer, displace the complementary DNA strand, move the polymerase along the template, and initiate the synthesis of RNA primers on the displaced strand. None of these reactions occur in the absence of gp32. The entire nature of this requirement is still undetermined; however, it is clear that only the presence of gp32 enables polymerase and accessory proteins to replicate templates with extensive secondary structure. Since neither gp32 nor the other individual proteins of the replication complex denature native DNA, we assume that the complex association of proteins at a replication fork provides additional sites on the DNA and sources of energy that facilitate helix melting and strand displacement. We may also have suggested that homologous DNA binding proteins are more important for lagging strand synthesis than for leading strand synthesis. We are unaware of any data that precisely answer this question.
Recovery of Bacteroides thetaiotaomicron ameliorates hepatic steatosis in experimental alcohol-related liver disease
Published in Gut Microbes, 2022
Moris Sangineto, Christoph Grander, Felix Grabherr, Lisa Mayr, Barbara Enrich, Julian Schwärzler, Marcello Dallio, Vidyasagar Naik Bukke, Archana Moola, Antonio Moschetta, Timon E. Adolph, Carlo Sabbà, Gaetano Serviddio, Herbert Tilg
DNA extraction from feces was performed by DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol. Following this, the amount of Bt DNA was quantified by qPCR with SybrGreen (Eurogentec, Köln, Germany) on MXPro3000 Cycler (Agilent Technology, Waldbronn, Germany). The primers for Bt DNA detection were based on 16rDNA gene sequences: forward-GGCAGCATTTCAGTTTGCTTG; reverse-GGTACATACAAAATTCCACACGT. The cycle was performed using 30 ng of fecal DNA, primer concentrations at 250 nM and 60°C as annealing temperature. The standard curve was obtained by serial dilutions of bacterial DNA extracted from Bt colonies, and the cycle threshold of each sample was compared with the standard. The quantity of Bt DNA in the feces was expressed in Log10 ng/g.
Improving genetic diagnostics of skeletal muscle channelopathies
Published in Expert Review of Molecular Diagnostics, 2020
Vinojini Vivekanandam, Roope Männikkö, Emma Matthews, Michael G. Hanna
First-generation genetic sequencing methods developed by Frederick Sanger in 1975 utilize RNA primers and labeled substrates to detect genetic mutations in families [22]. Although very accurate, it is a costly and time-consuming method. Next-generation sequencing (NGS), allowing parallel sequencing of DNA strands via a gene panel, has markedly improved diagnostic rates and times while simultaneously reducing cost [22]. This evolution of testing at the National Hospital for Neurology and Neurosurgery (NHNN) has improved diagnostic rates (Figure 2) which has led to more accurate prevalence estimates. Myotonia congenita, the most common of the muscle channelopathies, in 2019 has an estimated prevalence of 1.17 per 100 000, which is double the estimated prevalence prior to the implementation of next-generation sequencing in 2013.
An update on the biology and management of dyskeratosis congenita and related telomere biology disorders
Published in Expert Review of Hematology, 2019
Marena R. Niewisch, Sharon A. Savage
The end replication problem occurs due to the inability of DNA polymerases to completely replicate the telomeric C-rich lagging strand [37,38]. When the last RNA primer at the 3′ end of DNA is removed, the newly synthesized strand is a few nucleotides shorter and causes gradual telomere shortening. Hayflick and Moorhead were the first to report the replication limit of cells in culture, which is now known to occur due to progressive telomere shortening with each cell division [39]. When telomeres reach a critically short length, a non-replicative state known as cellular senescence is triggered and some cells may then undergo apoptosis [40]. Notably the shortest telomere on a single chromosome, not average telomere length, triggers the onset of cell senescence [41]. TERT expression is silenced in most human cells after the first few weeks of embryogenesis [42,43] and therefore, telomeres shorten during life and are a cellular marker of aging. Some stem cells such as germ cells, hematopoietic stem cells, expanding lymphocytes, skin cells and the intestinal lining continue to express telomerase and maintain telomeres at a constant length, evading cellular senescence [44–47].