Method to detect repeat sequence motifs in nucleic acid

Abstract

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.

1 .- 45 . (canceled) 
     
     
         46 . A primer pair for amplifying all or a portion of a CCG repeat tract in the 5′-untranslated region (5′-UTR) of fragile X related mental retardation gene 2 (FMR2), comprising an upstream primer and a downstream primer wherein:
 (i) the downstream primer comprises a CGG repeat segment which contains less than four consecutive CGG triplet repeats and hybridizes to the junction between the 3′ end of the CCG repeat tract in the FMR2 gene and sequence directly 3′ thereto; and/or 
 (ii) the upstream primer comprises a CCG repeat segment which contains less than four consecutive CCG triplet repeats and hybridizes to the junction between the 5′ end of the CCG repeat tract in the FMR2 gene and sequence directly 5′ thereto. 
 
     
     
         47 . The primer pair of  claim 46 , wherein the primer pair is able to determine the presence or absence of an expansion of the CCG repeat tract in the 5′-untranslated region (5′-UTR) of FMR2 in an individual. 
     
     
         48 . The primer pair of  claim 47 , wherein the expansion of the CCG tract is a full mutation or a premutation. 
     
     
         49 . The primer pair of  claim 46 , wherein the downstream primer comprises a second nucleic acid sequence with no sequence homology to the FMR2 gene, the second nucleic acid sequence located 5′ to the CGG triplet repeat sequence and the upstream primer comprises additional nucleic acid sequence with no sequence homology to the FMR2 gene, the additional nucleic acid sequence located 5′ to the CGG repeat sequence. 
     
     
         50 . The primer pair of  claim 46 , wherein the downstream primer further comprises a second nucleic acid sequence 5′ to the CGG triplet repeat sequence, the second nucleic acid sequence comprising sequence adjacent to the 3′ end of the CCG repeat tract in the FMR2 gene and the upstream primer further comprises a first additional nucleic acid sequence 5′ to the CCG repeat sequence, the first additional nucleic acid sequence comprising sequence adjacent to the 5′ end of the CCG repeat tract in the FMR2 gene. 
     
     
         51 . The primer pair of  claim 46 , wherein the downstream primer comprises two sets of two consecutive CGG triplets separated from each other by at least one nucleotide or the upstream primer comprises two sets of two consecutive CCG triplets separated from each other by at least one nucleotide. 
     
     
         52 . The primer pair of  claim 46 , wherein the upstream primer comprises a sequence selected from the group consisting of SEQ ID NOs: 14-40, 71-74, and 76. 
     
     
         53 . The primer pair of  claim 46 , wherein the downstream primer comprises a sequence selected from the group consisting of SEQ ID NOs: 41-69 and 77. 
     
     
         54 . The primer pair of  claim 46 , wherein the upstream primer comprises a sequence selected from the group consisting of SEQ ID NOs: 15-22 and 29-36. 
     
     
         55 . The primer pair of  claim 46 , wherein the downstream primer comprises a sequence selected from the group consisting of SEQ ID NOs: 42-49 and 56-66. 
     
     
         56 . The primer pair of  claim 46 , wherein the CGG repeat segment in the downstream primer is any one of the sequences selected from SEQ ID NOs: 83, 96-100, and 102-113. 
     
     
         57 . The primer pair of  claim 46 , wherein the CCG repeat segment in the upstream primer is any one of the sequences selected from SEQ ID NOs: 14 and 78-94. 
     
     
         58 . The primer pair of  claim 46 , wherein the downstream primer or the upstream primer is labeled with a detectable label. 
     
     
         59 . The primer pair of  claim 58 , wherein the detectable label is selected from fluorophores, radiolabel s, chemiluminiscent compound, colorimetric labels, magnetic labels, biotin, digoxigenin, haptens, proteins, or enzymes.