Method to detect repeat sequence motifs in nucleic acid

摘要

Methods for determining the presence or absence of expansion of CGG repeat sequence in the FMR1 gene presence or absence of expansion of CCG repeat sequence in the FMR2 gene are provided. The methods are useful in identifying an individual with normal/intermediate, versus premutation or full mutation allele of FMR1 gene and FMR2 gene due to the expansion of CGG repeats and CCG repeats in the 5′-untranslated region respectively. The methods are also useful for screening newborns for fragile X syndrome or for screening women to determine heterozygosity status with full premutation of the CCG repeat tract. The methods are also useful in estimating the premutation and full mutation carrier frequency and estimating the prevalence of FXTAS AND FXPOI in a population. The methods are simple, rapid and require small amount of sample.

主权项

1. A method for determining the presence or absence of an expansion of the CGG repeat tract in the 5′-untranslated region (5′-UTR) of fragile X related mental retardation 1 (FMRI) gene in an individual, said method comprising:
a) amplifying all or a portion of said 5′-UTR CGG repeat tract in the presence of a deoxy GTP analog, using as template genomic DNA comprising the FMR1 gene obtained from a biological sample from said individual, said amplifying being achieved with at least a primer pair, said primer pair comprising an upstream primer and a downstream primer, wherein:
(i) the downstream primer comprises a CCG repeat segment that comprises at least two CCG triplets and less than four consecutive CCG triplets, and hybridizes to the junction between the 3′ end of the CGG repeat tract in the FMR1 gene and sequence directly 3′ thereto, wherein said downstream primer consists essentially of a sequence selected from the group consisting of SEQ ID NOs 3, 23-26, 37-40, 73, and 74; or(ii) the upstream primer comprises a CGG repeat segment that comprises at least two CGG triplets and less than four consecutive CGG triplets, and hybridizes to the junction between the 5′ end of the CGG repeat tract in the FMR1 gene and sequence directly 5′ thereto, wherein said upstream primer consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 50-53 and 67-69, said amplifying generates amplicons that contain a portion of the CGG repeat tract; b) separating said amplicons according to size by capillary electrophoresis; and c) detecting the presence or absence of an expansion of the CGG repeat tract, wherein the size of said amplicons indicates the presence or absence of expansion of said CGG repeat tract in said 5′-UTR of FMRI gene.