Methods and compositions for the diagnosis of fragile X syndrome
AssigneeArch Development Corporation
InventorsSoma Das, David H. Ledbetter
Life of this patent
Estimating the term…
Abstract
The present invention relates generally to the field of diagnostics. More particularly, it concerns the use of methylation-specific PCR in order to identify those males having Fragile X syndrome. The present invention provides a method in which amplification specific for the methylated FMR1 sequence is observed in all individuals with a full mutation, while all normal and premutation individuals show only amplification specific for the unmethylated sequence, thus allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism also may detectable by this method. Thus, methylation-specific PCR is demonstrated as a rapid and reliable tool for the diagnosis of fragile X.
Claims24
A method for determining the methylation state of an FMR1 gene promoter of a male subject comprising the steps of:
- (a) denaturing a DNA sample from said subject;
- (b) subjecting the denatured DNA to bisulfite modification;
- (c) amplifying said DNA using primers pairs having the sequences
- ACCGATTCCCAACAACGCGCATA (SEQ ID NO:12) and
- TTTCGTTATCGTCGTCGTTCGC (SEQ ID NO:13), and
- ACACACATACACACACTCCCAAA (SEQ ID NO:14) and
- TTGAAATGGAGTTGAGTGTTTGAT (SEQ ID NO:15);
- and
- (d) detecting amplification products from step (c).
An isolated primer comprising the sequence
- ACCGATTCCCAACAACGCGCATA (SEQ ID NO:12).
An isolated primer comprising the sequence TTTCGTTATCGTCGTCGTTCGC (SEQ ID NO:13).
An isolated primer comprising the sequence
- ACACACATACACACACTCCCAAA (SEQ ID NO:14).
An isolated primer comprising the sequence
- TTGAAATGGAGTTGAGTGTTTGAT (SEQ ID NO:15).
A set of two primer pairs comprising the following sequences:
- ACCGATTCCCAACAACGCGCATA (SEQ ID NO:12) and
- TTTCGTTATCGTCGTCGTTCGC (SEQ ID NO:13), and
- ACACACATACACACACTCCCAAA (SEQ ID NO:14) and
- TTGAAATGGAGTTGAGTGTTTGAT (SEQ ID NO:15).
A kit comprising, in suitable container means, primer pairs comprising the following sequences:
- ACCGATTCCCAACAACGCGCATA (SEQ ID NO:12) and
- TTTCGTTATCGTCGTCGTTCGC (SEQ ID NO:13), and
- ACACACATACACACACTCCCAAA (SEQ ID NO:14) and
- TTGAAATGGAGTTGAGTGTTTGAT (SEQ ID NO:15).
Dependencies are inferred from the claim text.