Improved Fluorescent PCR-Based Assay for Sizing CGG Repeats at the FRAXA Locus

Houdayer, Claude; Lemonnier, A; Gérard, M.; Chauve, Cédric; Tredano, M; Tb, de Villemeur; Aymard, P; Bonnefont, Jean‐Paul; Feldmann, Delphine

doi:10.1515/cclm.1999.065

Cited by 20 publications

(14 citation statements)

References 19 publications

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“…17 DMSO and 7-deaza-dGTP have successfully been used for the screening of the Fragile X Syndrome in mentally handicapped children in combination with Expand Long Template PCR system (Roche) in determining the CGG repeat number in males and females for alleles from normal to premutation size range and the detection of full mutations in males. 18,19 In our case, the combination of these two reagents was unable to guarantee a specific amplification, probably due to the different condition in the Expand Long Template PCR system provided by Roche. These molecules, used either alone or in combination with both the Gold Taq polymerase (Applied Biosystems) and the Taq polymerase provided by Eppendorf (data not shown), gave exactly the same results, suggesting that the combination of the three additives is fruitful with different Taq polymerases.…”

Section: Discussionmentioning

confidence: 76%

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Musso

Bocciardi

Parodi

et al. 2006

The Journal of Molecular Diagnostics

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Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives-betaine, dimethyl sulfoxide, and 7-deaza-dGTP-was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

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Section: Discussionmentioning

confidence: 76%

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Musso

Bocciardi

Parodi

et al. 2006

The Journal of Molecular Diagnostics

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“…5 as silver staining, 11 inclusion of an ␣-32 P-labeled dNTP in the reaction, or hybridization with a radioactive or chemiluminescent probe. The use of betaine instead of DMSO to reduce secondary structures or the Expand Long Template PCR system (Roche Diagnostics) has been independently proposed in the analysis of repeat expansions, [12][13][14] often in conjunction with fluorescence PCR-based assays.…”

Section: Enhanced Pcr For Fmr1 Expansions 609mentioning

confidence: 99%

An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene

Saluto¹,

Brussino²,

Tassone³

et al. 2005

The Journal of Molecular Diagnostics

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Several diagnostic strategies have been applied to the detection of FMR1 gene repeat expansions in fragile X syndrome. Here, we report a novel polymerase chain reaction-based strategy using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany) and the osmolyte betaine. Repeat expansions up to ϳ330 CGGs in males and up to at least ϳ160 CGGs in carrier women could be easily visualized on ethidium bromide agarose gels. We also demonstrated that fluorescence analysis of polymerase chain reaction products was a reliable tool to verify the presence of premutation and full mutation alleles both in males and in females. This technique, primarily designed to detect premutation alleles, can be used as a routine first screen for expanded FMR1 alleles. (J Mol Diagn 2005, 7:605-612)Fragile X syndrome is the most common inherited form of mental retardation. This syndrome is caused by the expansion of CGG repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1) gene and hypermethylation of its 5Ј upstream CpG island.1 The CGG repeat element is polymorphic, varying from 6 to 44 repeats in the normal range, from 45 to 54 repeats in the gray zone, and from 55 to 200 repeats in the premutation range.2 For alleles below the gray zone, the CGG repeat is generally stable in parent-to-offspring transmissions. However, CGG elements in the premutation range become increasingly unstable with increasing repeat number, and alleles exceeding ϳ59 CGG repeats can expand to a full mutation in a single generation, almost exclusively by transmission from mother to son. The FMR1 premutation is typically associated with specific clinical manifestations unique to the premutation range: premature ovarian failure has been observed in ϳ20% of women, 3-6 whereas the fragile X-associated tremor/ ataxia syndrome has been found in at least one-third of carrier males more than 50 years old.7-9 Individuals affected with fragile X syndrome have FMR1 alleles with a CGG repeat number greater than 200.At present, DNA analysis of the CGG expansion is primarily performed using Southern blot analysis, which is able to detect alleles spanning the range from normal to large full mutation alleles; however, this method lacks the resolution to accurately size alleles. An alternative approach, using polymerase chain reaction (PCR) amplification of the region spanning the CGG repeat, provides much greater resolution, although it suffers from the difficulty of amplifying CGG repeats greater than ϳ100 to 150 repeats, because of the high GC content of the sequence being amplified.Radioactive or chemiluminescent probing, or fluorescence PCR, can overcome most problems, at least in the premutation range. Several studies have already described a number of PCR techniques, which use diverse combinations of DNA polymerase, 7-deaza-dGTP, and co-solvents such as dimethyl sulfoxide (DMSO) and betaine. 10 -15 However, the largest allele that has been amplified to date is 250 CGG repeats, 13 and PCR results with alleles of greater than ϳ...

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“…Fifty nanograms of DNA were amplified in a 20 l reaction mixture that contained 200 M each of dATP, dCTP, dTTP, and 7-deaza-2Ј-deoxy-GTP, 10 mM Tris-HCl, pH 8.3, 40 mM NaCl, 2 mM MgCl 2 , 0.25 U Taq Platinum (Life Technologies, Bethesda, MD), 0.5 M primers, and 10% DMSO. The DMSO and 7-deaza-2Ј-deoxy-GTP are helpful in the amplification and analysis of GC-rich regions (McConlogue et al, 1988;Houdayer et al, 1999). The thermocycling protocol was as follows: 5 min denaturing at 96°C, 35 cycles of 20 sec at 96°C, 20 sec at 68°C, and 40 sec at 72°C, and a 20-min…”

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confidence: 99%

Uncommon TGFBRI allele is not associated with increased susceptibility to colon cancer

Samowitz

Curtin

Leppert

et al. 2001

Genes Chromosomes & Cancer

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Previous studies have suggested that an allele of the transforming growth factor-beta type I receptor (TGFBRI) gene that codes for six instead of the usual nine alanines in a polyalanine repeat is associated with an increased susceptibility to colon cancer, and that the six-alanine homozygote is seen only in individuals with some form of cancer. We evaluated this TGFBRI polymorphism in a population-based sample of 252 individuals with colon cancer and 362 age- and gender-matched controls from the state of Utah. TGFBRI genotypes were determined by PCR amplification and length determination of the polyalanine repeat. In addition to the common nine-alanine (9A) allele, we identified six- (6A), eight- (8A), ten- (10A), eleven- (11A), and twelve-alanine (12A) TGFBRI alleles. 6A/9A heterozygotes were seen in similar percentages of colon cancer cases (18.3%) and controls (16.0%). 6A/6A homozygotes were slightly more common in controls than in colon cancer cases (1.4% vs. 0.8%), and none of the controls with the 6A/6A genotype had any of the non-colonic cancers reported in previous studies. We conclude that the 6A TGFBRI allele is not associated with an increased susceptibility to colon cancer at the population level, and that the 6A/6A homozygote is not restricted to individuals with some form of cancer.

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Improved Fluorescent PCR-Based Assay for Sizing CGG Repeats at the FRAXA Locus

Cited by 20 publications

References 19 publications

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene

Uncommon TGFBRI allele is not associated with increased susceptibility to colon cancer

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