Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader–Willi Syndrome and Duchenne Muscular Dystrophy

Hung, Chia-Cheng; Chen, Chih‐Ping; Lin, Shuan‐Pei; Chien, Shih‐Chang; Lee, Chien‐Nan; Cheng, Wen‐Fang; Hsieh, Wu‐Shiun; Liu, Ming S.; Su, Yi‐Ning; Lin, Win-Li

doi:10.1373/clinchem.2006.071118

Cited by 16 publications

(9 citation statements)

References 53 publications

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“…Moreover, the polymorphism in the proximal CMT-A-REP elements may lead to genetic misdiagnosis [29]. Competitive multiplex PCR for deletion and duplication analyses is a new technique that is currently in use [30][31][32][33]. It is easier and faster than traditional diagnostic approaches.…”

Section: Discussionmentioning

confidence: 99%

Identification of deletion and duplication genotypes of thePMP22gene using PCR‐RFLP, competitive multiplex PCR, and multiplex ligation‐dependent probe amplification: A comparison

et al. 2008

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We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.

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Section: Discussionmentioning

confidence: 99%

Identification of deletion and duplication genotypes of thePMP22gene using PCR‐RFLP, competitive multiplex PCR, and multiplex ligation‐dependent probe amplification: A comparison

et al. 2008

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“…20 It is easier and faster than the traditional diagnostic approaches. The principle involves three amplifications of a target gene and two reference genes in a single tube.…”

Section: Discussionmentioning

confidence: 99%

Quantitative and Qualitative Analyses of the SNRPN Gene Using Real-Time PCR with Melting Curve Analysis

Hung

Lin

et al. 2011

The Journal of Molecular Diagnostics

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“…The multiplex PCR protocol involves three amplifications of one target gene and two reference genes in a single tube [28,29]. Large deletions in the PWS-Angelman critical region at 15q11-q13 fragments can be easily identified; however, the limitation of the FISH and multiplex PCR is that only the deletion type of PWS/AS can be determined.…”

Section: Discussionmentioning

confidence: 99%

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

Hung

Lin

et al. 2009

Electrophoresis

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In this article, we show that methylation-specific multiplex PCR (MS-multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS-multiplex PCR to simultaneously amplify three sequences: the 3' ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation-sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader-Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS-multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS-multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in-house-designed MS-multiplex PCR protocol is a relatively simple, cost-effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.

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Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader–Willi Syndrome and Duchenne Muscular Dystrophy

Cited by 16 publications

References 53 publications

Identification of deletion and duplication genotypes of thePMP22gene using PCR‐RFLP, competitive multiplex PCR, and multiplex ligation‐dependent probe amplification: A comparison

Identification of deletion and duplication genotypes of thePMP22gene using PCR‐RFLP, competitive multiplex PCR, and multiplex ligation‐dependent probe amplification: A comparison

Quantitative and Qualitative Analyses of the SNRPN Gene Using Real-Time PCR with Melting Curve Analysis

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

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