A Single Polymerase Chain Reaction-Based Protocol for Detecting Normal and Expanded Alleles in Myotonic Dystrophy

Gennarelli, Massimo; Pavoni, M.; Amicucci, Paola; Novelli, Giuseppe; Dallapiccola, Bruno

doi:10.1097/00019606-199806000-00002

Cited by 26 publications

(16 citation statements)

References 8 publications

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“…For genes with trinucleotide repeats that expand as dynamic mutations, the alleles in the normal range are easily amplified by PCR, but there is a possibility that larger alleles are not amplified, as is the case in myotonic dystrophy (Gennarelli et al, 1998), resulting in an apparently homozygous genotype. Nevertheless, the CAG expanded repeat has been easily diagnosed by PCR (Brice, 1998).…”

Section: Discussionmentioning

confidence: 99%

The SCA1 (Spinocerebellar ataxia type 1) and MJD (Machado-Joseph disease) CAG repeats in normal individuals: segregation analysis and allele frequencies

2003

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Spinocerebellar ataxia type 1 (SCA1) and Machado-Joseph disease (MJD/SCA3) are autosomal dominant neurodegenerative diseases caused by expansions of a CAG trinucleotide repeat in the SCA1 and MJD genes. These expanded sequences are unstable upon transmission, leading to an intergeneration increase in the number of repeats (dynamic mutation). The transmission of the CAG repeat was studied in normal mother-father-child trios, referred for paternity testing (SCA1, n = 367; MJD, n = 879). No segregation distortion was detected. The CAG allele frequencies were determined in 330 unrelated individuals (fathers from couples tested for paternity). The allele frequency distributions did not differ from those previously reported for European populations. The estimated values for the statistic parameters indicating diversity at the SCA1 locus did not differ much from those reported previously for other STRs in the Brazilian population, while those for the MJD locus were close to or higher than the maximum values of previous reports. This shows that SCA1 and MJD are highly informative loci for applications in genetic and population studies and for forensic analysis.

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Section: Discussionmentioning

confidence: 99%

The SCA1 (Spinocerebellar ataxia type 1) and MJD (Machado-Joseph disease) CAG repeats in normal individuals: segregation analysis and allele frequencies

2003

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“…This could be confirmed only with the help of Southern blot analysis, by which the presence of large unamplifiable alleles can be excluded. As many as 25% unaffected individuals are homozygous for normal MD1 alleles (Brook et al, 1992;Gennarelli et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Many scientific groups have already worked on a simple, reproducible protocol to amplify the CTG-repeat expansion within the DMPK gene (Cheng et al, 1996;Gennarelli et al, 1998). The DMPK gene regions are GC rich and tend to form certain secondary structures.…”

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confidence: 99%

New Analysis Method of Myotonic Dystrophy 1 Based on Quantitative Fluorescent Polymerase Chain Reaction

Skrzypczak-Zielińska

Sulek²,

Mierzejewski³

et al. 2009

Genetic Testing and Molecular Biomarkers

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Molecular genetic testing of myotonic dystrophy type 1 (DM1) is based on the identification and determination of a cytosine-thymine-guanine (CTG) repeat expansion in the DMPK gene. This is usually done by Southern blot analysis-a time-consuming and very laborious technique requiring high molecular weight DNA. The aim of our study was to develop a highly sensitive, rapid, and cost-effective molecular analysis characterizing the CTG repeat region of the DMPK gene based on a two-step polymerase chain reaction (PCR) protocol. (1) For the detection of alleles of up to 100 repeats, a quantitative fluorescent (QF) amplification with primers flanking the repeat region of the DM1 locus and two reference genes (PAX2 and DHCR7) for standardization was used. By this method it was possible to identify both homozygous and heterozygous DM1 alleles. (2) Long PCR was only performed if a single wild-type allele was detected that gave a QF-PCR signal of only half intensity compared to a homozygous sample. The results obtained using combined QF and Long PCR are highly accurate compared with Southern blot analysis. We conclude that our new rapid analysis is reliable for genetic testing of DM1 patients.

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“…This technique requires specific conditions to amplify large fragments with the appropriate normal and affected samples followed by the addition of an end labelled probe [15]. This process is similar to Triplet Primed-PCR in the case that duplicated or deleted repeats in a sequence may remain undetectable.…”

Section: Southern Blotting Of Long Range Pcr Productsmentioning

confidence: 99%

Myotonic Dystrophy Type 1 Diagnostics: A Changing Trend

Singh¹

2013

IOSR-JPBS

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A Single Polymerase Chain Reaction-Based Protocol for Detecting Normal and Expanded Alleles in Myotonic Dystrophy

Cited by 26 publications

References 8 publications

The SCA1 (Spinocerebellar ataxia type 1) and MJD (Machado-Joseph disease) CAG repeats in normal individuals: segregation analysis and allele frequencies

The SCA1 (Spinocerebellar ataxia type 1) and MJD (Machado-Joseph disease) CAG repeats in normal individuals: segregation analysis and allele frequencies

New Analysis Method of Myotonic Dystrophy 1 Based on Quantitative Fluorescent Polymerase Chain Reaction

Myotonic Dystrophy Type 1 Diagnostics: A Changing Trend

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