Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.
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http://dx.doi.org/10.1016/0378-4347(95)00370-3 | DOI Listing |
Nucleic Acids Res
January 2025
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Maidashi 3-1-1, Higashi-Ku, Fukuoka 812-8582, Japan.
An enzyme with strong single-stranded DNA (ssDNA) ligation activity would be advantageous for many molecular biology applications. However, currently available enzymes exhibit only limited activity. Here, we identified an enzyme with strong ssDNA ligation activity upon searching the databases for proteins homologous to TS2126 RNA ligase, the known enzyme with the highest yet limited ssDNA ligation activity.
View Article and Find Full Text PDFJ Virol
January 2025
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Unlabelled: Coronaviruses have large, positive-sense single-stranded RNA genomes that challenge conventional strategies for mutagenesis. Yeast genetics has been used to manipulate large viral genomes, including those of herpesviruses and coronaviruses. This method, known as transformation-associated recombination (TAR), involves assembling complete viral genomes from dsDNA copies of viral genome fragments via homologous recombination in .
View Article and Find Full Text PDFAnal Chem
January 2025
School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China.
Formamidopyrimidine DNA glycosylase (Fpg) and flap endonuclease 1 (FEN1) are essential to sustaining genomic stability and integrity, while the abnormal activities of Fpg and FEN1 may lead to various diseases and cancers. The development of simple methods for simultaneously monitoring Fpg and FEN1 is highly desirable. Herein, we construct a multiple cyclic ligation-promoted exponential recombinase polymerase amplification (RPA) platform for sensitive and simultaneous monitoring of Fpg and FEN1 in cells and clinical tissues.
View Article and Find Full Text PDFCancer Res
January 2025
INSERM U1194, Montpellier Cedex 05, Occitanie, France.
BRCA1 deficiency is observed in approximately 25% of triple-negative breast cancer (TNBC). BRCA1, a key player of homologous recombination (HR) repair, is also involved in stalled DNA replication fork protection and repair. Here, we investigated the sensitivity of BRCA1-deficient TNBC models to the frequently used replication chain terminator gemcitabine, which does not directly induce DNA breaks.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Achieving targeted hypermutation of specific genomic sequences without affecting other regions remains a key challenge in continuous evolution. To address this, we evolved a T7 RNA polymerase (RNAP) mutant that synthesizes single-stranded DNA (ssDNA) instead of RNA in vivo, while still exclusively recognizing the T7 promoter. By increasing the error rate of the T7 RNAP mutant, it generates mutated ssDNA that recombines with homologous sequences in the genome, leading to targeted genomic hypermutation.
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