A Brief Practical Guide to PCR McKinzie, Page B; Myers, Meagan B
Methods in molecular biology (Clifton, N.J.),
2023, Volume:
2621
Journal Article
Polymerase chain reaction (PCR) has been a powerful molecular biology tool since the mid-1980s. Millions of copies of specific sequence regions of DNA can be generated to allow the study of these ...regions. Fields that use this technology range from forensics to the experimental study of human biology. Standards for performing PCR and information tools to help design PCR protocols aid in successful implementation of PCR.
Molnupiravir (MOV) is used to treat COVID‐19. In cells, MOV is converted to the ribonucleoside analog N4‐hydroxycytidine (NHC) and incorporated into the SARS‐CoV‐2 RNA genome during its replication, ...resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS‐CoV‐2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow‐frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E. coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome‐wide mutation frequencies in a dose‐dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract
Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for ...quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10−8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10−4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10−5 to 10−1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive base read was evaluated to determine if there is a limit of read length for quantitation of ≥10−4, and it was determined that read lengths up to 70 bases are reliable for quantitation. The method described here is adaptable to various oncogene or tumor suppressor gene targets, with the potential to implement multiplexing at the initial tagging step. While easy to perform manually, it is also suited for robotic handling and batch processing of samples, facilitating detection and quantitation of genetic carcinogenic biomarkers before tumor formation or in normal-appearing tissue.
DNA base editors (BEs) composed of a nuclease‐deficient Cas9 fused to a DNA‐modifying enzyme can achieve on‐target mutagenesis without creating double‐strand DNA breaks (DSBs). As a result, BEs ...generate far less DNA damage than traditional nuclease‐proficient Cas9 systems, which do rely on the creation of DSBs to achieve on‐target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off‐target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10−8 mutations per base pair. In this proof‐of‐principle study, we evaluated whether this technique could also detect the on‐ and off‐target mutations generated by a cytosine‐to‐thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on‐target mutant allele fractions ranging from ~7% to ~63%, depending on the single‐guide RNA (sgRNA) used, while no on‐target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3‐fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on‐ and off‐target mutations in cell populations that have undergone genome editing.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Background
Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin‐induced DNA damage, however, may also ...result in mutations in normal cells and the initiation of secondary malignancies. In the current study, we have used the erythrocyte PIG‐A assay to evaluate mutagenesis in non‐tumor hematopoietic tissue of cancer patients receiving cisplatin chemotherapy.
Methods
Twenty‐one head and neck cancer patients undergoing treatment with cisplatin were monitored for the presence of PIG‐A mutant total erythrocytes and the young erythrocytes, reticulocytes (RETs), in peripheral blood for up to five and a half months from the initiation of the anti‐neoplastic chemotherapy.
Results
PIG‐A mutant frequency (MF) in RETs increased at least two‐fold in 15 patients at some point of the monitoring, while the frequency of total mutant RBCs increased at least two‐fold in 6 patients. A general trend for an increase in the frequency of mutant RETs and total mutant RBCs was observed in 19 and 18 patients, respectively. Only in one patient did both RET and total RBC PIG‐A MFs did not increase at any time‐point over the monitoring period.
Conclusion
Cisplatin chemotherapy induces moderate increases in the frequency of PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy.
Cisplatin chemotherapy induces moderate increases in the frequency of PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T‐cells of rats treated in vivo with N‐propyl‐N‐nitrosourea (PNU) or procarbazine (PCZ). The signatures ...from the treated rats were different from the signature of background mutations. The main component of the spontaneous T‐cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU‐induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ‐induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10–40× increases in the number of mutations per clone were detected in T‐cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X‐linked Pig‐a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such ...assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome‐wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error‐free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome‐wide ultralow‐frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10−7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23‐fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4 h, and 5‐, 12‐, and 29‐fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4 h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS‐exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA‐containing sample, including those that are not compatible with conventional mutation detection approaches.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•The entire genome of the cell line L5178Y-3.7.2C (TK+/−) is sequenced.•Whole genome analysis reveals millions of mutations and genetic markers.•The genome of L5178Y-3.7.2C (TK+/−) harbors large ...homozygous deletions.
The mouse lymphoma L5178Y-3.7.2C (TK+/−) cell line is extensively used in genetic toxicology to conduct the mouse lymphoma assay (MLA). The MLA is used to establish the mutagenic and clastogenic effects of chemicals and pharmaceuticals, and is one of the few genetic tests widely accepted by regulatory agencies throughout the world. Despite the extensive use and regulatory impact of L5178Y-3.7.2C (TK+/−) cells, little is known about their genetic composition or how it affects the outcome of the MLA. To determine the genetic background of this cell line, we sequenced and analyzed its entire genome. Our results confirm the existence of previously described mutations in the Tk1 and Trp53 genes and catalog millions of other mutations, many of which impair the function of genes with key roles in cell physiology and genetic toxicology.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig‐a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to ...analyze nucleated bone‐marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig‐a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig‐a mutation and loss of GPI anchors was further strengthened and the genesis of Pig‐a mutation in PCZ‐dosed rats was evaluated by analyzing bone‐marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48‐deficient mutant phenotype bone‐marrow granulocytes (BMGs CD11b+) were isolated by flow‐cytometric sorting. Sequencing data showed that the CD48‐deficient mutant phenotype BMGs contained mutations in the Pig‐a gene while wild‐type BMGs did not. PCZ‐induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ‐induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig‐a assay and mutation in the Pig‐a gene. Also, PCZ mutation spectra were similar in bone‐marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig‐a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic ...treatment with vancomycin.
The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates.
The seven isolates (MEH1-MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1β.
We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.
PDF