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At least 649 records · Page 36Linked to original sources
PubMed2001

Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination.

Large deletions in the dystrophin gene account for > 60% of mutations responsible for Duchenne muscular dystrophy (DMD). We have developed a genetic test that can be used directly for the preimplantation genetic diagnosis (PGD) of a majority of couples at risk of transmitting DMD. The test, a double nested multiplex polymerase chain reaction assay for the amplification of exons 8, 19, 45, 47 and 51 allows the detection of over 70% of all DMD deletions. Amelogenin sequences on the X and the Y chromosomes were also co-amplified to provide a correlation between embryo gender and deletion status. The setting up of reliable single cell assays for preimplantation genetic diagnosis is delicate and time consuming. Assays have to be validated on a large number of single cells for each specific mutation to assess efficiency and accuracy before being applied clinically. The multiplex procedure permitted the validation of all tested loci in the same series of isolated lymphocytes rather than in separate series for each exon. One hundred single lymphocytes, 50 female and 50 male cells, were analysed with an overall amplification rate of 98% and an amplification failure of 2% per exon. We suggest that this test is reliable, easy to set up and much preferable to a mere sex determination with the selective transfer of female embryos.

Dystrophin↗
PubMed1992

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis.

Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.

Amino Acid Sequence↗
PubMed2001

Development of a multiplex PCR for the detection of Haplosporidium nelsoni, Haplosporidium costale and Perkinsus marinus in the eastern oyster (Crassostrea virginica, Gmelin, 1971).

Haplosporidium costale (SSO), Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo) have caused oyster mortality on the North American east coast since the 1950s. Currently, the monitoring of oyster populations for these pathogens depends on histopathology for H. nelsoni, H. costale and the Ray/Mackin assay for P. marinus. In this study we describe the development and optimization of a multiplex polymerase chain reaction (MPCR) for the detection of H. nelsoni, H. costale and P. marinus. In addition, we determine its specificity and sensitivity. The MPCR clearly detects and differentiates the protozoan pathogens. There was no cross-reactivity between these species. The MPCR was able to detect DNA from H. nelsoni as low as 10 fg and P. marinus and H. costale as low as 1 pg. The MPCR allows for the detection of H. nelsoni, H. costale and P. marinus in a single PCR reaction.

Animals↗
PubMed1994

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines.

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2 bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.

Base Sequence↗
PubMed2004

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

Acute Disease↗
PubMed2002

Multiplex PCR for simultaneous detection of 677 C-->T and 1298 A-->C polymorphisms in methylenetetrahydrofolate reductase gene for population studies of cancer risk.

Methylenetetrahydrofolate reductase (MTHFR) plays a pivotal role in folate metabolism by regulating the diversion of folate metabolites toward DNA methylation or toward DNA synthesis. Because aberrations in both of these pathways can be tumor promoting, the two common polymorphisms in the MTHFR gene, 677 C-->T and 1298 A-->C, have been implicated as risk factors for several cancers. Homozygosity for the 677 C-->T polymorphism and compound heterozygosity for 677 C-->T and 1298 A-->C polymorphisms both reduce enzyme activity by more than 50% and can promote oncogenic alterations in DNA methylation especially when folate status is low. Thus, rapid identification of both polymorphisms in MTHFR gene would be of importance in understanding the genetics of abnormal folate metabolism as related to human cancer risk. Here we describe a multiplex polymerse chain reaction/restriction fragment length polymorphism procedure in which two sets of primers are used to amplify simultaneously the DNA regions spanning 677 and 1298 loci in one PCR reaction. The amplified products are digested by HinfI or MboII followed by agarose gel electrophoresis for simultaneous detection of the 677 C-->T and 1298 A-->C polymorphisms in the same gel.

Base Sequence↗
PubMed2004

Genetic characterization of Chinese hereditary non-polyposis colorectal cancer by DHPLC and multiplex PCR.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease due to germline mutations of human mismatch repair genes, mainly hMLH1 and hMSH2. The aim of the present study was to identify the point mutations and large genomic deletions of hMLH1 and hMSH2 genes in 14 Chinese HNPCC families. METHODS: Fourteen families fulfilling the Chinese HNPCC criteria were involved in this study. Genomic DNA isolated from peripheral blood samples was analyzed. Point mutations were detected by denaturing high performance liquid chromatography (DHPLC) followed by DNA sequencing. Multiplex polymerase chain reaction and GeneScan analysis were employed to detect the large genomic deletions of these two genes. RESULTS: Four of the 14 probands (29%) had sequence abnormalities that probably affect the protein function in the exonic regions of hMLH1 and hMSH2 genes. Included were one complete deletion of exons 1-7 and one missense mutation of the hMSH2 gene, and one nonsense mutation and one missense mutation of the hMLH1 gene. The large genomic deletion accounted for 25% (one out of four) of all mutations. Half (two out of four, 50%) of the mutations were missense mutation. In addition, one silent mutation, four polymorphisms in the exonic regions and four polymorphisms in the intronic regions were also discovered. CONCLUSIONS: Point mutations and large genomic deletions of the hMLH1 and hMSH2 genes were responsible for nearly one-third of Chinese HNPCC families. Detection of large genomic deletions should be involved in the routine screening manual for HNPCC families.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Single-tube multiplex PCR and capillary electrophoresis for the detection of bcl2-IgH chimeras.

The molecular background of follicular lymphoma is the formation of chimera(s) by the bcl2 gene and one of the immunoglobulin genes, mainly the heavy chain gene. While the diagnosis of follicular lymphoma is based on histological study, the histology cannot be recommended for the follow-up of efficiency of treatment(s). Sensitive detection of the chimera genes in blood or bone marrow samples can serve as informative data without invasive sampling. In this study, a single-tube multiplex reaction was developed for the simultaneous detection of different chimeras of the bcl2 and immunoglobulin heavy chain genes, molecular hallmarks of follicular lymphoma. The method was optimized for the highest yield of reaction and for reproducing the ratio of components in artificial mixtures of two different chimeras. The method is suitable for fast and accurate molecular characterization of follicular lymphomas and for follow-up of residual disease.

Chimera↗
PubMed2002

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination.

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test for D. nodosus. A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.

Animals↗
PubMed2004

Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray.

We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

Chromosomes, Human, Y↗
PubMed2006

Gross SDHB deletions in patients with paraganglioma detected by multiplex PCR: a possible hot spot?

Pheochromocytoma and paraganglioma are rare neuroendocrine tumors that arise in the adrenal medulla and the extra-adrenal paraganglia, respectively. Inheritance of these tumors is mainly a result of mutations affecting the VHL, RET, NF1, and SDH genes. Germ-line mutations of the SDH genes have been found to account for nearly 10% of apparently sporadic cases. Nevertheless, alterations other than point mutations have not yet been well characterized. In this study, we investigated the frequency of gross SDH deletions in 24 patients who tested negative for point mutations and had at least one of the recommended features for genetic testing. For this purpose, we used a technique that is easy to implement in the lab to specifically detect gross deletions affecting SDHB, SDHC, and SDHD. We identified 3 heterozygous SDHB deletions (3/24) in 3 independent cases with paraganglioma: 1 whole SDHB deletion and 2 deletions exclusively affecting exon 1. These latter mutations match the unique gross deletion previously reported, indicating this region could be a hot spot for gross SDHB deletions. It seems likely that these alterations can account for a considerable number of both familial and apparently sporadic paraganglioma cases. Although this is the first report describing the presence of gross deletions in patients with apparently sporadic paragangliomas, the extra-adrenal location of the tumor seems to constitute a determining factor for whether to include these patients in genetic testing for gross deletions in the SDHB gene.

Adolescent↗
PubMed2006

Semiquantitative multiplex PCR: a useful tool for large rearrangement screening and characterization.

Methods presently employed for detection of large rearrangements have several drawbacks, such as the amount of sample and time required, technical difficulty, or the probability of false-negative carriers. Using the low-density-lipoprotein receptor (LDLR) gene, whose mutations are responsible for familial hypercholesterolemia (FH), we have developed a procedure to detect large rearrangements in this gene based on semiquantitative PCR, with important improvements as compared to previous methods. Our method covers the complete LDLR gene and introduces an internal control in the reaction. The procedure discriminates the four different large rearrangements (two deletions and two insertions) that we have used as positive mutation controls (Valencia-1 to -5). All altered exons from each rearrangement are identified. Furthermore, when families from probands carrying these large rearrangements (34 members) were analyzed, our results agreed with those obtained previously with Southern blot. We have also analyzed a sample of 110 unrelated FH probands and the method has correctly identified the two different large rearrangements present and insertions or deletions as small as 1 bp. In conclusion, the method we present allows the identification of large rearrangements affecting exons of the gene, including small insertions or deletions or complete gene deletion. In addition, it constitutes a first characterization step of rearrangements, and is easy to carry out fast, and can be applied to the analysis of any gene.

Apolipoproteins B↗
PubMed1993

Analysis of 6 VNTR loci by 'multiplex' PCR and automated fluorescent detection.

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to 'allelic dropout' was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.

Alleles↗
PubMed2004

Simultaneous determination of matrix metalloproteinase (MMP)-7, MMP-1, -3, and -13 gene expression by multiplex PCR in colorectal carcinomas.

BACKGROUND AND AIMS: MMP-7, a member of the matrix metalloproteinase family, is believed to play a significant role in the growth and proliferation of colon cancer cells. The aim of this study was to evaluate MMP-7 gene expression in comparison with MMP-1, MMP-3, and MMP-13 in patients with resectable rectal and colon cancer by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Biopsy samples of tumor ( n=30) and distant normal mucosa ( n=30) from 30 patients were obtained intraoperation. Messenger (m)RNA was extracted from all of the tissue samples and reverse transcribed to double-stranded cDNA. Semi-quantitative RT-PCR was performed to study the MMP gene expression in both the tumor and normal mucosal specimens. MMP mRNA values were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample. RESULTS: In all 30 cases an increase in MMP-7 mRNA expression was detected in the cancerous tissue ( p=0.00004). In 21 out of 30 cases an increase in MMP-13 mRNA ( p=0.023) and in 22 out of 30 cases an increase in MMP-3 mRNA ( p=0.075) was detected in the cancerous tissue. In contrast, there was no significant change in the MMP-1 expression of normal and cancerous mucosal specimens in either colon or rectal carcinomas. There were no significant differences between rectum and colon carcinomas. CONCLUSION: Taking into account our earlier studies, we conclude that most cases of colorectal carcinogenesis are characterized by enhanced expression of MMP-7, -13, -3 and the gelatinases, whereas MMP-1-expression is very inconsistent and not overexpressed in many cases. MMP-7 inhibition as well as inhibition of MMP-13 and MMP-3 may be a useful preventive or therapeutic adjunct in colorectal cancer.

Aged↗