Join the Resolution

Asuragen AmplideX FMR1 PCR reagents are a state-of-the-art research tool which eliminates the use of the Southern Blot.

FMR1 genes with >200 CGG repeats are usually hypermethylated and inactive, resulting in loss of FMRP, an important protein involved in microRNA and mRNA regulation and neural development.

Consequences

The consequence of this gene inactivation was revealed in a seminal centre study (Rousseau et al) in 1994 which demonstrated the the prediction of intellectual disablilty was improved by considering both the CGG expansion size and the gene methylation status. Individuals with a greater than 200 CGG but unmethylated or mosaic allele had a normal cognitive phenotype or less severe phenotype. The severity of fragile X syndrome symptoms in females can also vary based on the methylation status of the allele.

Southern Blot

Since '91 the primary method used to determine methylation status of the allele has been Southern blot. Southern blot is laborious, has low resolution and sensitivity and requires large quantities of high-quality DNA compared to PCR-based methods. Determining the methylation status without Southern blot have relied on either chemical or enzymatic pretreatment of the DNA before PCR analysis.

Southern Blot method

Chemical methods

Chemical methods require sodium bisulfite to convert methylated cytosine to uracil. These changes can be recognized through various techniques including PCR, DNA sequencing and melting curve analysis. These methods are more suitable to analyse male samples because of the presence of two X chromosomes in female samples can co-found the analysis. Enzyme based methods use methylation sensitive restriction enzymes to digest the unmethylated DNA. Real-time PCR, probe ligation and/or capillary electrophoresis can or may be used to determine the methylation sequence by comparing amplicons generated from digested and undigested DNA.

Recently, it was shown that enzymatic pretreatment and full-length amplification of the CGG repeats can yield repeat length and methylation status in a high troughput workflow which is accurate for both male and female samples. Emerging markers in the FMR1 gene region may also be predictive of the fragile X phenotype. These novel capabilities have the potential to improve the methylation assesment, and eliminate the need for Southern blot analysis.

 

source: http://www.nomoresouthernblots.com/

 

AmplideX FMR1 PCR Reagents

35 years

We use cookies to ensure that we give you the best experience on our website. More information

Accept