Chryslaine Rodríguez-Tanty

Synthesis of 5-Methyl-2′-O-Deoxycytidine Analogs to Determine Monoclonal Antibody Specificity in the Recognition of the 6-(p-Bromobenzoylamino) Caproyl Radical

Abstract Eight new p-bromobenzoyl derivatives of 5-methyl-2′-O-deoxycytidine analogs, substituted at the 4-position, were synthesized. The best conditions for obtaining 5-methyl-4-N-aminoalkyl-2′-O-deoxycytidine from 3′,5′-di-O-acetyl-4–(1,2,4-triazol-1-yl)-2′-O-deoxythymidine were studied. The nucleoside analogs were used to identify the fragment of the 6-(p-bromobenzoylamino)caproyl radical that binds to the monoclonal antibody obtained against it and to define an affinity scale of monoclonal antibody against them.

Comparative Study of Three Methods for Non-radioactive In Vivo DNA Labeling in Escherichia coli Using Nucleoside Analogs

Abstract In the present work, a comparative study of 5-FdUrd, thy -, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5α cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labelled DNA (MDLD) was 312 pg with the 5-FdUrd and thy - methods for 5-BrdUrd labelled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy - and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.

DNA-labelled cytidine assay for the quantification of CAG repeats

The sequencing procedure has been used to determine the size of the CAG repeat expansion for the diagnosis of genetic disorders. Likewise, standard polymerase chain reaction (PCR) and gel electrophoresis techniques are applied for screening large number of patients. The trinucleotide repeats (TNR) region amplification by means of the PCR procedure was initially performed using 32-P end-labelled primers and currently carried out with fluorescently end-labelled primers. The goal to obtain reliable TNR quantification assays, at low cost and short assay times, represents a challenge for the molecular diagnosis aimed at massive screening of affected populations. In the current work, we obtained preliminary results of a new methodology for the detection and size estimation of CAG expanded alleles. The assay was based on an indirect enzyme linked immunosorbent assay (ELISA) for quantifying the amount of labelled cytidines in DNA molecules. The label, 6-(p-bromobenzamido)caproyl radical, was introduced by the transamination and acylation reactions. A group of model sequences containing different numbers of CAG repeats, as well as the ATXN3 (ataxin 3) gene (from subjects suffering type 3 spinocerebellar ataxia SCA3) were used for assay standardization. The assay is simple, inexpensive, and easy to perform and differentiates distinct degrees of CAG expansions.

Non-enzymatic in vitro DNA labeling and label immunoquantification.

Abstract In the present work, a label immunoquantification procedure was developed in order to determine the number of markers introduced into DNA. A non‐enzymatic, in vitro labeling method for introducing the p‐bromobenzoyl radical (label), through transamination and acylation reactions of the cytidine nucleotides in calf thymus DNA, was carried out. Three spacer arms with different lengths were used for separating the label from the nucleotide and three labeled DNA were obtained. Anti‐p‐bromobenzoyl chicken IgY polyclonal antibodies were obtained. The antibodies detected the label, into three‐labeled DNA, with different sensitivities, in relation to spacer arm length used. About 3–11 labels per 4 × 106 bases into thermally denatured DNA were immunoquantified.