Methods and reagents for detecting and assessing genotoxicity
A genomic and pharmaceutical technology, applied in all aspects of this technology in the field of preclinical and clinical medicine, the unique mutation field of each double base pair, can solve problems such as nucleic acid mutation, birth defects, damage, etc.
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example 1
[0239] Double sequencing in Application of mouse cII transgenes and in vivo mutation analysis in endogenous genes. This section describes an example where error-corrected next-generation sequencing (NGS) was used to directly measure Chemically induced mutations in the cII transgene and native mouse genes used in the transgenic rodent (TGR) mutation assay. Currently, TGR mutation assays detect rare cII mutations through plaque formation. Standard NGS because of its high error rate (every 10 3 bases sequenced ~ 1 error) cannot be used for low-frequency mutation detection. Error-corrected NGS, or duplex sequencing, has a significantly lower error rate (~1 / 10 8 bases), allowing detection of ultra-rare mutations.
[0240] In this example, the application of dual sequencing was used to assess the effects of control, N-ethyl-N-nitrosourea (ENU), and benzo[a]pyrene (B[a]P) exposures. Mutation frequency (MF) and spectrum in C57BL6 male mice.
[0241] On days 1-28, with vehicl...
example 2
[0254] Direct quantification of in vivo chemical mutagenesis in mammalian genomes using dual sequencing. This section describes an example where duplex sequencing was used to determine whether early mutations in cancer driver genes reflect the tumorigenic potential of a test mutagen.
[0255] In this example, the effect of urethane was examined in different mouse tissue types (lung, spleen, blood) in an FDA-approved cancer-prone mouse model: Tg.rasH2 (Saitoh et al. Oncogene 1990 .PMID2202951). This mouse contains ~3 tandem copies of human Hras with an activating enhancer mutation to promote expression on one hemizygous allele. These mice are susceptible to splenic angiosarcoma and lung adenocarcinoma, and are routinely used in 6-month carcinogenicity studies as an alternative to 2-year natural animal studies. Tumors found in mice often have acquired activating mutations in one copy of the human Hras proto-oncogene. In addition to the 4 native mouse genes (Rho, Hp, Ctnnbl, P...
example 3
[0280] Analysis of mutagen signatures in mammalian genomes using duplex sequencing. This section describes an example where data generated from a duplex sequencing analysis can be used to generate and compare mutagenic signatures for mutagen identification and / or to identify mutagen exposure.
[0281] The Catalog of Somatic Mutations in Cancer (COSMIC) database provides a reference for "mutation signatures," which are defined as unique combinations of mutation types found in a genome. Somatic mutations are present in all cells of the body and occur throughout life. Such somatic mutations are, for example, the result of multiple mutational processes involving intrinsic slight infidelity of the DNA replication machinery, exposure to exogenous or endogenous mutagens, enzymatic modification of DNA and defective DNA repair.
[0282] Figures 17A-17C is showing the flag 1 from COSMIC ( Figure 17A ), flag 4 ( Figure 17B ) and sign 29 ( Figure 17C A plot of the mutation spectr...
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