scholarly journals Improvement in Sensitivity of Allele-specific PCR Facilitates Reliable Noninvasive Prenatal Detection of Cystic Fibrosis

2004 ◽  
Vol 50 (4) ◽  
pp. 694-701 ◽  
Author(s):  
Ourania Nasis ◽  
Shanel Thompson ◽  
Tom Hong ◽  
Margaret Sherwood ◽  
Shawn Radcliffe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Alterations ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Specific Pcr ◽  
Wild Type Sequence ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Type Sequence

Abstract Background: Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. Methods: We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11–19.2 weeks), with mutation confirmation by chorionic villus sampling. Results: The method detected 5 copies of the CF D1152H mutant allele in the presence of up to ∼100 000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. Conclusions: This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.

Detection of Imatinib-Resistant BCR-ABL Mutants in Drug-Naïve Patients: Correlation with Disease Phase and Clonal Evolution but Not with Response to Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 273-273
Author(s):  
Michael W. Deininger ◽  
Stephanie Willis ◽  
Thoralf Lange ◽  
Shannon McWeeney ◽  
Sandra Otto ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Clonal Evolution ◽  
Drug Binding ◽  
Disease Stage ◽  
Wild Type ◽  
Imatinib Resistance ◽  
Specific Pcr ◽  
Blastic Phase ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: Point mutations in the kinase domain (KD) of BCR-ABL that impair drug binding are a common cause of imatinib resistance. In some cases the mutations present at relapse were detected in pretherapeutic samples, suggesting selection in the presence of imatinib and a possible association with disease progression. To test this hypothesis, we studied the presence of KD mutations in imatinib-naïve patients in various disease phases. Methods and patients: we developed fluorescent allele-specific RT-PCR assays for 8 common KD mutations (Q252H, Y253F, Y253H, E255K, E255V, T315I, M351T, F359V), comprising approximately 75% of mutations detected in patients, using universal BCR and mutation-specific ABL primers. Mutation-complementary oligonucleotides were further modified to favor amplification of mutant over wild type. Sensitivity and specificity were optimized in serial dilutions of mutant in wild type, using plasmid DNA or cDNA extracted from BaF3 cells expressing BCR-ABL constructs. The assays reproducibly detected mutations with a sensitivity of at least 10−4 – 10−5. To avoid false-positive results cDNA was prepared in a physically separate lab that never handled recombinant mutant BCR-ABL. Negative controls included “blanks”, K562 cells (positive for wild type BCR-ABL) and HL60 cells (BCR-ABL-negative). Pretherapeutic samples from 20 patients in first chronic, 24 in accelerated and 21 in blastic phase were studied. Results: Mutations were detected in 13 samples from blastic phase (5) or from accelerated phase (8) but in none from chronic phase. Mutations were: T315I (7), Y253F (2), 253H (2), Q252H (1) and F359V (1). Sequencing of the same material detected F359V in one sample (consistent with the allele specific PCR), and K247R in another (positive for T315I by allele-specific PCR) but was wild type in the remaining 10 samples with good quality readings. Multivariate analysis of baseline factors revealed significant and independent correlations between mutation detection and disease phase (P = 0.0008) and clonal cytogenetic evolution (P = 0.0004). In contrast, mutation detection was not an independent predictor of failure to achieve complete hematologic response (CHR) or major cytogenetic response (MCR) and was not correlated with overall survival. Four patients with mutations achieved CHR and 3 MCR; 4 patients failed to achieve CHR, 1 died from neutropenic sepsis and 1 discontinued because of skin toxicity. Thus far, follow-up specimens during imatinib therapy have been sequenced in 6 patients. In 2 cases (T315I and Q252H) the identical mutation was detected, one case (T315I) showed K247R (detected prior to therapy by sequencing but not included in the allele specific assays), and 3 cases were wild type BCR-ABL. Conclusion: We provide direct evidence that the presence of BCR-ABL KD mutantions is correlated with disease stage. Moreover, the strong association with clonal evolution suggests that genomic instability may be responsible for both phenomena. However, not all mutants that were detected prior to therapy were subsequently selected in the presence of imatinib, suggesting that some mutations may occur in cells without self-renewal capacity or that additional factors may be required to confer a fully resistant phenotype.

Novel Assay for the Identification of NOTCH1 PEST Domain Mutations Using Fragment Analysis and Allele Specific PCR

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5127-5127
Author(s):  
Paulo Vidal Campregher ◽  
Roberta Cardoso Petroni ◽  
Nair Muto ◽  
Rubia Santana ◽  
Roberta Sitnik ◽  
...  
Keyword(s):  
Cost Effective ◽  
Single Mutation ◽  
Wild Type ◽  
Double Peak ◽  
Fragment Analysis ◽  
Specific Pcr ◽  
Routine Identification ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Notch1 Mutations

Abstract Abstract 5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c. 7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34. We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c. 7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs: A single 391 bp peak: wild type samplesThree peaks (391 bp, 389 bp and 356 bp): heterozygous for c. 7544_7545delCTTwo peaks (391 bp and another bigger or smaller, depending on the size of insertion/deletion): another insertion or deletion, but not c. 7544_7545delCT. We have studied 46 de-identified blood samples from patients with CLL, in several diverse stages, using our assay. In 40 patients, there was no NOTCH1 mutation detected. Six patients had a pattern compatible with c. 7544_7545delCT NOTCH1 mutation (see figure 1), and no patients presented with another mutation. Overall the frequency of NOTCH1 mutations in our series was 13 %. Selected mutated samples were confirmed through amplicon sequencing. In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations using fragment analysis and allele specific pcr that is suitable for implementation in the clinical setting for CLL patients evaluation. We will continue testing more CLL patients in order to identify another, rarer, NOTCH1 mutations. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations . / A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. . / B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A super-suppressor on the thirteenth linkage group in Saccharomyces

1966 ◽  
Vol 8 (1) ◽  
pp. 1-7
Author(s):  
Yasuji Oshima ◽  
Ikue Oshima
Keyword(s):  
Linkage Group ◽  
Mechanism Of Action ◽  
Auxotrophic Mutant ◽  
Wild Type Allele ◽  
Wild Type ◽  
Linkage Groups ◽  
Type Allele ◽  
Allele Specific ◽  
Linkage Relationships

It has been proved that the suppression of seven different auxotrophic mutant alleles is brought about by a single super-suppressor allele designated S. Super-suppressor S is allele-specific rather than locus-specific and is dominant to its wild-type allele.Suppressible mutant alleles show no linkage relationships either among themselves or with the suppressor S.Super-suppressor S has been shown to be closely linked (about 5 stranes) to one of the centromeres other than those of linkage groups I to XII. This establishes a new thirteenth linkage group.The possible mechanism of action of the super-suppressor is discussed.

scholarly journals Limits of Dideoxysequencing in the Detection of Somatic Mutations in Gastrointestinal Stromal Tumors

2015 ◽  
Vol 15 (3) ◽  
pp. 13-20 ◽  
Author(s):  
K Jasek ◽  
V Buzalkova ◽  
P Szepe ◽  
L Plank ◽  
Z Lasabova
Keyword(s):  
Gastrointestinal Stromal Tumors ◽  
Somatic Mutations ◽  
Low Cost ◽  
Gene Mutations ◽  
Wild Type ◽  
Stromal Tumors ◽  
Specific Pcr ◽  
Allele Specific ◽  
Detection Of Mutations ◽  
Allele Specific Pcr

Abstract Detection of mutations in cancer is particularly important in terms of proper treatment and targeted therapy. The aim of this study was the comparison of two methods: allele-specific PCR (AS-PCR) and dideoxysequencing applied for the identification of BRAF gene mutations in wild-type gastrointestinal stromal tumors (WT GISTs). We have optimized the conditions for the detection V600E mutation representing the c.1799 T>A substitution by AS-PCR and have used dideoxysequencing to verify our results. In nine cases, we were able to detect the mutation by AS-PCR approach; however, the mutations have been confirmed by dideoxysequencing in four cases only. AS-PCR is fast and low cost method for the detection of V600E mutation which was validated as a sensitive assay for the identification of the most common BRAF mutation in DNA extracted from paraffin-embedded tissue of WT GISTs.

A novel genotyping technique for discriminating LVAS-associated hotspot mutations in SLC26A4 gene

2020 ◽  
Author(s):  
Chen Zhou ◽  
Xiangman Zou ◽  
Cuiying Peng ◽  
Guoqiang Gao ◽  
Zifen Guo
Keyword(s):  
Ct Scan ◽  
Primer Extension ◽  
Wild Type Allele ◽  
Wild Type ◽  
Specific Primers ◽  
Mutant Type ◽  
Type Allele ◽  
Slc26a4 Gene ◽  
Allele Specific ◽  
Hotspot Mutations

Abstract An increasing number of biological and epidemiological evidence suggests that IVS7-2A>G and 2168A>G mutations of solute carrier family 26, member 4 ( SLC26A4 ) gene plays a critical role in the development of large vestibular aqueduct syndrome (LVAS). In this study, we developed a rapid genotyping method for discriminating LVAS-associated hotspot mutations in SLC26A4 gene. The genotyping technique consists of 3’ terminal exonuclease-resistant phosphorothioate-modified allele specific primer extension mediated by exo + polymerase. In PCR amplification by Pfu polymerase, allelic specific primers perfectly matching wild type allele were extended while no specific products were yielded from primers targeting mutant type allele. Similarly, allelic specific primers perfectly matching mutant type allele were extended and no specific products were observed from primers targeting wild type allele . The clinical application of 3’ terminal phosphorothioate-modified allele specific primers extension mediated by Pfu polymerase identified both homozygous for SLC26A4 gene IVS7-2A>G mutation in two LVAS patients diagnosed as by temporal bone CT scan. The genetic results from this method is consistent with that of DNA sequencing. The data suggest that exo + polymerase-mediated 3’ terminal phosphorothioate-modified primer extension is reliable in the identification of SLC26A4 gene hotspot mutation prior to high resolution CT scan. The method is extremely suitable for quickly molecular etiologic screening and early diagnosis and aggressive prevention therapy of LVAS.

SNP Chip Analysis of Myelodysplastic Syndromes Disclosed High Frequency of Uniparental Disomy and a Novel Dominant Mutation as the Target of 11q UPD.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 119-119
Author(s):  
Masashi Sanada ◽  
Lee-Yung Shih ◽  
Takahiro Suzuki ◽  
Go Yamamoto ◽  
Yasuhito Nannya ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
High Frequency ◽  
Copy Number ◽  
Uniparental Disomy ◽  
Wild Type Allele ◽  
Wild Type ◽  
Dominant Mutation ◽  
Type Allele ◽  
Allele Specific ◽  
Homozygous Mutations

Abstract Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by ineffective hematopoiesis and high propensity to leukemias. One of the prominent features of MDS is the high frequency of copy number changes that result in genetic imbalances. Loss of heterozygosity (LOH) may be also closely related to the pathogenesis of MDS, but genome-wide LOH mapping of MDS is still unexplored. We developed a robust program, CNAG/AsCNAR, to explore genetic alterations of the cancer genome, using Affymetrix® GeneChip® SNP arrays. Implementing a series of data compensation algorithms, it enables accurate copy number estimation in cancer genomes. In addition, by sensing subtle distortions in allele-specific signals caused by allelic imbalance using anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers even in the face of up to 70% normal cell contamination. Using GeneChip with CNAG/AsCNAR analysis, we analyzed a total of 172 MDS and MDS/MPD specimens in order to identify relevant potential target genes for development of MDS. Nnumerous copy number alterations were sensitively detected in MDS genomes, however more interesting finding was the high frequency of copy number neutral LOH (Uniparental disomy, UPD) observed in 51 of 172 (∼30%) MDS cases. They preferentially involved 1p, 1q, 4q, 7q, 11q, 17p and other chromosomal segments, which were associated with homozygous mutations of tumor suppressor genes and oncogenes, including TP53 (17p UPD), AML1/RUNX1 (in 21q UPD), Nras and cMPL (1p UPD), JAK-2 (9p UPD), and FLT3 (13q UPD). In addition we identified the target of 11q UPD, internally called mds11, which showed homozygous mutations in 8 of 9 MDS cases with 11q UPD (CMML=5, RAEB=3, overt leukemia=1). Mutations of mds11 were rare in non-11q UPD cases (1/163), demonstrating the mutation is tightly linked to 11q UPD. NIH-3T3 cells transduced with these mutatnts showed increased colony formation in soft agar and were tumorigenic in nude mice. To investigate the functions of these mutants in hematopoietic cells, we introduced this mutant gene into c-kit(+)Sca1(+)Lin(−) murine bone marrow cells, and examined their replating capacities in methylcellurose culture, where all the mutants showed enhanced capacities. These observations also present a novel examples that duplication of a dominant mutation (11qUPD) with exclusioin of the wild-type allele is an important mechanism of tumorigenesis. In conclusion, UPD is an important mechanism of development of MDS, in which both gain-of-function and loss-of-function mutations are duplicated with exclusion of wild-type allele. Analysis of 11q UPD disclosed novel gain-of-function mutations. Identification of the targets of UPDs in 1q, 4q and 7q should also be important to gain a novel insight into the pathogenesis of MDS.

Allele specific PCR with microfluorometry: application to the detection of del F508 mutation in cystic fibrosis

2002 ◽  
Vol 316 (1-2) ◽  
pp. 147-154 ◽  
Author(s):  
Akihiro Yamaguchi ◽  
Juan Alberto Nepote ◽  
Maliheh Kadivar ◽  
Yasuko Tagami ◽  
Masaru Fukushi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr
Sign in / Sign up

Export Citation Format

Share Document