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1.
Anal Biochem ; 419(2): 161-7, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-21925477

ABSTRACT

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.


Subject(s)
Real-Time Polymerase Chain Reaction/methods , Temperature , Angelman Syndrome/diagnosis , Angelman Syndrome/genetics , Humans , Prader-Willi Syndrome/diagnosis , Prader-Willi Syndrome/genetics
2.
Clin Chem ; 56(7): 1098-106, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20472822

ABSTRACT

BACKGROUND: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. METHODS: The assay is based on combining methylation-sensitive FastDigest(R) endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. RESULTS: With our novel approach, we correctly diagnosed the imprinting disorders Prader-Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay's accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. CONCLUSIONS: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.


Subject(s)
Angelman Syndrome/diagnosis , DNA Methylation , DNA/analysis , Prader-Willi Syndrome/diagnosis , snRNP Core Proteins/genetics , Angelman Syndrome/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA Restriction Enzymes/chemistry , Feasibility Studies , Gene Dosage , Genetic Loci , Genome, Human , Genomic Imprinting , Humans , Polymerase Chain Reaction/methods , Prader-Willi Syndrome/genetics , Promoter Regions, Genetic
3.
Invest Ophthalmol Vis Sci ; 44(2): 493-6, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12556373

ABSTRACT

PURPOSE: To compare the changes with increasing age of ERG parameters in relation to clinical data in two distinct phenotypes of genetically determined, dominantly inherited macular drusen: malattia leventinese (ML) and Zermatt macular dystrophy (ZMD). METHODS: Ganzfeld rod- and cone-electroretinograms (ERGs) from 15 patients affected with ML and 14 patients with ZMD and clinical data were analyzed retrospectively. The patients' ages ranged from 20 to 77 years in the ML group and from 9 to 74 years in the ZMD group. RESULTS: Both inherited macular degenerations caused a marked decrease in visual acuity, the latest after age 65. Most patients with ML retained good visual function (0.8-1.0) until the fifth decade, followed by a rapid decrease in the fifth or sixth decade. ZMD is characterized by a relatively continuous decrease in visual acuity with increasing age. Morphologically, in the juvenile stages in both entities, drusen were observed at the posterior pole. Rod-driven and cone-driven ERG b-wave amplitudes decreased nearly linearly in ML and ZMD in accord with the normal loss of amplitude with increasing age. Implicit times of cone b-waves for ML increased markedly with age, whereas in ZMD the values were always prolonged beyond the normal range with a slight increase with age. CONCLUSIONS: In terms of visual acuity, the progression of both dominantly inherited macular dystrophies is quite different. This is not reflected in the amplitudes of the b-waves in the Ganzfeld ERGs, which decrease normally for both entities. Implicit times of the cone-b waves were more markedly prolonged in ML compared with ZMD. In-depth longitudinal documentation of the natural course of those dominantly inherited macular diseases should facilitate patient counseling.


Subject(s)
Retina/physiology , Retinal Drusen/genetics , Retinal Drusen/physiopathology , Visual Acuity/physiology , Adolescent , Adult , Aged , Aging/physiology , Child , Cross-Sectional Studies , Dark Adaptation , Disease Progression , Electroretinography , Humans , Middle Aged , Retinal Drusen/classification , Retrospective Studies
4.
Klin Monbl Augenheilkd ; 219(4): 206-10, 2002 Apr.
Article in German | MEDLINE | ID: mdl-12022002

ABSTRACT

BACKGROUND: To show the value of Ganzfeld electroretinography (ERG) in Malattia Leventinese (ML, or Hereditary Dominant Drusen) and Zermatt Macular Dystrophy (ZMD) and to illustrate multifocal electroretinography (mfERG) in 2 cases of ML. PATIENTS AND METHODS: In 15 patients with ML and 14 with ZMD we recorded Ganzfeld ERGs along with clinical examinations. In two patients with ML, and an we also performed a mfERG and an automated and Goldmann perimetry. All patients had a genotypic confirmation of the respective disease. For ERG measurements, the UTAS-3000 system was used, the mfERG was recorded using the RetiScan system. RESULTS: In ML, the visual acuity remained at 0.8 or higher until the 5 (th) or 6 (th) decade of life, followed by a rapid drop. In ZMD, the decrease in acuity began already in the 3 (rd) decade and followed a more continuous time course. The time course of the decrease of the ERG b-wave amplitudes was nearly identical for either disease. The mfERG showed in one case of ML a marked reduction in the macular response density but, in the second case, a normal density response pattern despite large degenerative changes at the posterior pole. In both of these patients, we found no visual field defects. CONCLUSIONS: Patient history and clinical testing raised the suspicion of a hereditary macular dystrophy. By means of Ganzfeld and multifocal electroretinography the course of the disease could be observed. However, definite diagnosis could only be established by genetic identification.


Subject(s)
Corneal Dystrophies, Hereditary/diagnosis , Electroretinography/methods , Adult , Age Factors , Aged , Aged, 80 and over , Chromosome Aberrations , Corneal Dystrophies, Hereditary/genetics , Diagnosis, Differential , Female , Genes, Dominant , Humans , Male , Middle Aged , Signal Processing, Computer-Assisted , Visual Acuity/physiology , Visual Field Tests/methods
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