Use of dual-tags for the evaluation of genomic variable repeat regions

A method for evaluating the length of a (CGG), trinucleotide repeat region within an FMR1 gene, said method comprising:

• (a) amplifying a (CGG)n trinucleotide repeat region within an FMR1 gene with a primer pair, each primer of the primer pair comprising a target specific sequence, a differentiating tag sequence 5′ of the target specific sequence and a blocker between the target specific sequence and the differentiating tag sequence to produce a dual-tagged amplification product;
• (b) labeling the dual-tagged amplification product;
• (c) digesting the dual-tagged amplification product with a restriction enzyme to produce a (CGG)n trinucleotide repeat region fragment and an internal control fragment;
• (d) hybridizing the trinucleotide repeat region fragment to a first capture complex comprising an anti-tag sequence and a solid support, and hybridizing the internal control fragment to a second capture complex comprising an anti-tag sequence and a solid support;
• (e) detecting a signal produced by the label on the immobilized trinucleotide repeat region fragment and a signal produced by the label on the internal control fragment;
• and
• (f) comparing the intensity of the signal of the (CGG)n trinucleotide repeat region fragment and the signal of the internal control fragment to evaluate the length of the (CGG), trinucleotide repeat region within the FMR1 gene.

A method for diagnosing a trinucleotide expansion disease on the X chromosome comprising:

• (a) obtaining a nucleic acid sample from a subject to be tested;
• (b) amplifying a target nucleic acid sequence in the sample with a primer pair, each primer of the primer pair comprising a target specific sequence, a differentiating tag sequence 5′ of the target specific sequence and a blocker between the target specific sequence and the differentiating tag sequence to produce a dual-tagged amplification product;
• (c) labeling the dual-tagged amplification product;
• (d) digesting the dual-tagged amplification product with a restriction enzyme to produce an expansion region fragment and an internal control fragment;
• (e) hybridizing the expansion region fragment to a first capture complex comprising an anti-tag sequence and a solid support, and hybridizing the internal control fragment to a second capture complex comprising an anti-tag sequence and a solid support;
• (f) detecting a signal produced by the label on the immobilized expansion region fragment and a signal produced by the label on the internal control fragment;
• and
• (g) comparing the intensity of the signal of the expansion region fragment and the signal of the internal control fragment to diagnose a trinucleotide expansion disease if the intensity of the signal of the expansion region fragment is above a pre-determined threshold value.

A method for diagnosing Fragile-X syndrome comprising:

• (a) obtaining a nucleic acid sample from a subject to be tested;
• (b) amplifying a (CGG), expansion region within an FMR1 gene in the nucleic acid sample with a primer pair, each primer of the primer pair comprising a target specific sequence, a differentiating tag sequence 5′ of the target specific sequence and a blocker between the target specific sequence and the differentiating tag sequence to produce a dual-tagged amplification product;
• (c) labeling the dual-tagged amplification product;
• (d) digesting the dual-tagged amplification product with a restriction enzyme to produce an expansion region fragment and an internal control fragment;
• (e) hybridizing the expansion region fragment to a first capture complex comprising an anti-tag sequence and a solid support, and hybridizing the internal control fragment to a second capture complex comprising an anti-tag sequence and a solid support;
• (f) detecting a signal produced by the label on the immobilized expansion region fragment and a signal produced by the label on the internal control fragment;
• and
• (g) comparing the intensity of the signal of the expansion region fragment and the signal of the internal control fragment to diagnose Fragile-X syndrome if the intensity of the signal of the expansion region fragment is above a pre-determined threshold value.

A kit for evaluating a trinucleotide repeat region on the X chromosome comprising:

• (a) a primer pair capable of amplifying a trinucleotide repeat region on the X chromosome, each primer of the primer pair comprising a target specific sequence, a differentiating tag sequence 5′ of the target specific sequence and a blocker between the target specific sequence and the differentiating tag sequence;
• (b) a label;
• (c) a pair of capture complexes, each comprising an anti-tag sequence and a solid support;
• and
• (d) a restriction enzyme capable of cleaving an amplification product produced by said primer pair into a trinucleotide repeat region fragment and an internal control fragment.