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.
Pérez-Bello
Dannelys
D
Molecular Biology Department, Cuban Neuroscience Center, La Havana, Cuba.
Xu
Z H
ZH
Higginson-Clarke
David
D
Rojas
Ana María Riverón
AM
Le
Weidong
W
Rodríguez-Tanty
Chryslaine
C
D000067699:Ataxin-3; D001483:Base Sequence / Q000235:genetics; D001681:Biological Assay / Q000379:methods; D003562:Cytidine / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005821:Genetic Techniques; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics; D020754:Spinocerebellar Ataxias / Q000175:diagnosis / Q000235:genetics*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D003562:Cytidine; D004247:DNA; C092341:ATXN3 protein, human; D000067699:Ataxin-3