Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset
Maryam SedghiNayereh NouriEsmat Fazel-NajafabadiMansoor SalehiMajid HosseinzadehMahdieh BehnamMohammadreza Ghazavi
17
Citation
24
Reference
10
Related Paper
Citation Trend
Abstract:
Background: The Duchenne muscular dystrophy (DMD) gene is located in the short arm of the X chromosome (Xp21). It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA) over multiplex polymerase chain reaction (PCR) assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.Keywords:
Multiplex
Objective To evaluate the application of fluorescent multiplex amplification of short tandem repeat(STR)for monitoring survival of engraftment after bone marrow transplantation(BMT).Methods Three STR loci named D12S391,D18S865 and D20S161 in 56 cases were detected by fluorescent multiplex amplification.PCR products were separated and typed by DNA Sequencer.Results The genotypes of STR in 52 recipient after bone marrow transplantation were completely identical with those of the donors.In another 4 cases the evidences of mixed chimerism were observed.Conclusion The system of fluorescent multiplex amplification of STRs exhibited high capacity of discrimination and low cost.Its application in the detection of STR after BMT is reliable,sensitive and simple.Combined with the clinical manifestation it can be used to evaluate the effect of BMT.
Multiplex
Cite
Citations (0)
Multiplex
Cite
Citations (12)
Background: The Duchenne muscular dystrophy (DMD) gene is located in the short arm of the X chromosome (Xp21). It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA) over multiplex polymerase chain reaction (PCR) assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.
Multiplex
Cite
Citations (17)
Multiplex
Cite
Citations (59)
Multiplex
Cite
Citations (62)
The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used for analysis of copy number variations (CNVs) in single or multiple loci. MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis. Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions. However, an increasing demand for new gene specific assays makes it necessary to self-design new MLPA probes for which the available software may not be applicable. During evaluation of new self-designed reference probes, we encountered a number of problems, especially when applying the MLPA methodology to tumor samples.DNA samples from 48 unaffected individuals and 145 breast cancer patients were used to evaluate 11 self-designed MLPA probes and determine the cut-off values for CNV, before applying the MLPA probes to normalize the target probes in a cohort of affected individuals. To test the calculation strategy, three probes were designed to cover regions in Regulator of G-protein Signaling 8 (RGS8), which we previously have identified as being affected by allelic imbalance by LOH analysis across RGS8 in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.Here, we present a straightforward method, based upon the normalization pattern in both unaffected and affected individuals, to evaluate self-designed reference probes and to calculate CNV for the MLPA assay with specific focus on the difficulties when analyzing tumor DNA.
Multiplex
Cite
Citations (3)
By simultaneously amplifying several loci in the same reaction, multiplex PCR has been used in gene mapping and DNA typing with polymorphic short tandem repeat loci. Previous studies have discussed in detail the various parameters and conditions that influence the quantity of individual products generated by multiplex PCR. In practice, when a primer pair fails to amplify in a multiplex PCR for some individuals, singleplex PCR is often employed as a supplement to amplify the primer pair. However, the reliability of this procedure is unknown. In this study, we used six primer pairs from ABI PRISMTM Linkage Mapping Set version 2 to perform multiplex and singleplex reactions. The fluorescence-labeled amplification products were separated and detected on ABI PRISM 310 Genetic Analyzer. We found that for the marker D1S468, multiplex and singleplex reactions for the majority of individuals yielded reactions of different sizes. Therefore, the potential size difference between multiplex and singleplex reactions needs to be investigated. This investigation is essential to employ multiplex PCR supplemented with singleplex PCR in gene mapping and DNA typing.
Multiplex
Primer (cosmetics)
STR multiplex system
Cite
Citations (18)
We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common α‐thalassaemia deletions and one α‐globin gene triplication. The new assay detects the α 0 deletions – – SEA , – (α) 20.5 , – – MED , – – FIL and – – THAI in the first multiplex PCR, the second multiplex detects the –α 3.7 deletion and ααα anti3.7 variant, the third multiplex detects the –α 4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where α‐thalassaemias are prevalent.
Multiplex
Cite
Citations (364)
Multiplex ligation-dependent probe amplification (MLPA) is a multiplex copy number analysis tool which is routinely used to detect large mutations in genetic diseases. With continuous modifications, MLPA has been extended for the detection of DNA methylation variation, single nucleotide polymorphisms, chromosome abnormalities and other forms of genomic variation. The combination with other techniques has even enlarged the application of MLPA in molecular diagnostics of various human diseases. In this review, the principle of MLPA-based techniques as well as their main and latest applications in clinical detection are described. It is believed that with improved automation, increased multiplexing, lower cost and the combination with other technologies, MLPA will play an increasingly important role in molecular diagnosis of human disease.
Multiplex
Molecular diagnostics
Cite
Citations (8)
Recent improvements in the multiplex ligation-dependent probe amplification (MLPA) method promise successful multiplex analysis of various genetic markers. In particular, it has been demonstrated that elimination of the stuffer sequence included in MLPA probes for length-dependent analysis substantially simplifies the probe design process and improves the accuracy of the analysis. As is the case for other CE-based methods, MLPA could be further developed on a microchip platform. However, high-resolution analysis of short MLPA probes requires careful microchip operation. In this study, we developed a microchip device for the multiplex analysis of five food-borne pathogens using a stuffer-free probe set. Microchip channel design and electrophoresis operating conditions were first optimized for reproducible analysis, after which two sieving matrices were tested. Finally, the method was validated using DNA samples isolated from intentionally infected milk.
Multiplex
Cite
Citations (7)