scholarly journals High-Throughput Mutation Detection Method to Scan BRCA1 and BRCA2 Based on Heteroduplex Analysis by Capillary Array Electrophoresis

2004 ◽  
Vol 50 (2) ◽  
pp. 313-320 ◽  
Author(s):  
Eva Esteban-Cardeñosa ◽  
Mercedes Duran ◽  
Mar Infante ◽  
Eladio Velasco ◽  
Cristina Miner
Keyword(s):  
Single Strand Conformation Polymorphism ◽  
Heteroduplex Analysis ◽  
Breast Cancer Patients ◽  
Brca1 And Brca2 ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Coding Regions ◽  
Hands On ◽  
Dna Sequence Variants ◽  
Capillary Array

Abstract Background: Scanning for mutations in BRCA1 and BRCA2 in a large number of samples is hampered by the large sizes of these genes and the scattering of mutations throughout their coding sequences. Automated capillary electrophoresis has been shown to be a powerful system to detect mutations by either single-strand conformation polymorphism or heteroduplex analysis (HA). Methods: We investigated the adaptation of gel-based HA of BRCA1 and BRCA2 to a fluorescent multicapillary platform to increase the throughput of this technique. We combined multiplex PCR, three different fluorescent labels, and HA in a 16-capillary DNA sequencer and tested 57 DNA sequence variants (11 insertions/deletions and 46 single-nucleotide changes) of BRCA1 and BRCA2. Results: We detected all 57 DNA changes in a blinded assay, and 2 additional single-nucleotide substitutions (1186 A>G of BRCA1 and 3624 A>G of BRCA2), previously unresolved by conformation-sensitive gel electrophoresis. Furthermore, different DNA changes in the same PCR fragment could be distinguished by their peak patterns. Conclusions: Capillary-based HA is a fast, efficient, and sensitive method that considerably reduces the amount of “hands-on” time for each sample. By this approach, the entire coding regions of BRCA1 and BRCA2 from two breast cancer patients can be scanned in a single run of 90 min.

scholarly journals Structure and Temporal Dynamics of Populations within Wheat Streak Mosaic Virus Isolates

2001 ◽  
Vol 75 (21) ◽  
pp. 10231-10243 ◽  
Author(s):  
Jeffrey S. Hall ◽  
Roy French ◽  
T. Jack Morris ◽  
Drake C. Stenger
Keyword(s):  
Mosaic Virus ◽  
Temporal Dynamics ◽  
Consensus Sequence ◽  
Single Strand Conformation Polymorphism ◽  
Wheat Streak Mosaic Virus ◽  
Relative Fitness ◽  
Nucleotide Sequencing ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Constant Rate

ABSTRACT Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G6016) in NIa that was restored to A6016 in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.

scholarly journals Characteristics of BRCA-associated breast cancer in the population of the Russian Federation

2021 ◽  
Author(s):  
EI Novikova ◽  
EA Kudinova ◽  
VK Bozhenko ◽  
VA Solodkiy
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Russian Federation ◽  
False Negative ◽  
Brca2 Gene ◽  
Breast Cancer Patients ◽  
Brca1 And Brca2 ◽  
Coding Regions ◽  
Brca1 And Brca2 Genes ◽  
The Russian Federation

"Standard" diagnostic panels allow identification of only a few of BRCA1 and BRCA2 gene mutations most common in a population. Therefore, tests relying on such panels may return false negative results, since the coding regions of these genes may have other defects. For breast cancer (BC) patients, false negative test results may translate into selection of inadequate therapy by their doctors. This study aimed to identify the features of BRCA-associated breast cancer in the population of the Russian Federation. The study included breast cancer patients (n = 4440). At the first stage, all patients were screened for the eight most common BRCA1 and BRCA2 genes mutations with the help of real-time PCR. Next, patients that exhibited clinical signs of a hereditary disease (CSHD) in the absence of common mutations (n = 290) had the entire coding regions of BRCA1 and BRCA2 genes studied with next generation sequencing (NGS). "Standard" mutations in the BRCA1 and BRCA2 genes were identified in 169 (3.8%) cases. In the CSHD group, such mutations were revealed in 15.4% of cases. NGS uncovered 33 rare pathogenic BRCA1 and BRCA2 gene mutations in 40 out of 290 breast cancer patients (13.8%). It was concluded that among the residents of the Russian Federation, the range of pathogenic variants of BRCA-associated breast cancer is wide, and it stretches beyond the mutations considered by the "standard" diagnostic panels. Analysis of the entire coding regions of BRCA1 and BRCA2 genes allows increasing efficiency of detection of germline mutations in breast cancer patients at least twofold.

scholarly journals Nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Joanna Katarzyna Strzelczyk ◽  
Anna Slemp-Migiel ◽  
Magdalena Rother ◽  
Karolina Gołąbek ◽  
Andrzej Wiczkowski
Keyword(s):  
Candida Albicans ◽  
Sequence Analysis ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Clinical Isolates ◽  
Primary Target ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Conformation Polymorphism

One of the mechanisms of Candida albicans resistance to azole drugs used in antifungal therapy relies on increased expression and presence of point mutations in the ERG11 gene that encodes sterol 14α demethylase (14DM), an enzyme which is the primary target for the azole class of antifungals. The aim of the study was to analyze nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates. The Candida albicans isolates represented a collection of 122 strains selected from 658 strains isolated from different biological materials. Samples were obtained from hospitalized patients. Fluconazole susceptibility was tested in vitro using a microdilution assay. Candida albicans strains used in this study consisted of two groups: 61 of the isolates were susceptible to azoles and the 61 were resistant to azoles. Four overlapping regions of the ERG11 gene of the isolates of Candida albicans strains were amplified and sequenced. The MSSCP (multitemperature single strand conformation polymorphism) method was performed to select Candida albicans samples presenting genetic differences in the ERG11 gene fragments for subsequent sequence analysis. Based on the sequencing results we managed to detect 19 substitutions of nucleotides in the ERG11 gene fragments. Sequencing revealed 4 different alterations: T495A, A530C, G622A and A945C leading to changes in the corresponding amino acid sequence: D116E, K128T, V159I and E266D. The single nucleotide changes in the ERG11 gene did not affect the sensitivity of Candida albicans strains, whereas multiple nucleotide substitutions in the ERG11 gene fragments indicated a possible relation with the increase in resistance to azole drugs.

scholarly journals Breast Cancer Associated Mutations Reported Among Malaysian Malay, Chinese and Indian Patients

2017 ◽  
Vol 1 (2) ◽  
Author(s):  
Weng Kit Yow ◽  
Rozi Mahmud ◽  
King Hwa Ling ◽  
Karuppiah Thilakavathy
Keyword(s):  
Breast Cancer ◽  
Tumour Suppressor Genes ◽  
Nucleotide Polymorphisms ◽  
Cancer Genes ◽  
Breast Cancer Patients ◽  
Brca1 And Brca2 ◽  
Single Nucleotide ◽  
Research Findings ◽  
Malaysian Chinese ◽  
Increased Activity

Breast cancer among women has reached an alarming stage compared to 20 years ago.  It was found that single nucleotide polymorphisms (SNPs) play an important role in the development of breast cancer.  SNPs may suppress or activate the functional genes, which then leads to an increased survival of cells due to the suppression of tumour suppressor genes or increased activity in detrimental proteins, resulting in cancers.  Recently, breast cancer research is given significant attention due to the increasing number of breast cancer patients in Malaysia each year.  However, breast cancer researches in Malaysia are mainly focused on well known breast cancer genes such as BRCA1 and BRCA2.  There is less emphasis on the other breast cancer contributing genes.  This paper was aimed to review the research findings on mutations associated with breast cancer in Malaysian Malay, Malaysian Chinese and Malaysian Indian as it has never been cumulatively reported and to ascertain the common mutations found in these three major Malaysia ethnicity.

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

Prevalence of BRCA1 and BRCA2 mutations in breast cancer patients from Cuba

2008 ◽  
Vol 7 (3) ◽  
pp. 275-279 ◽  
Author(s):  
Rolando Comacho Rodriguez ◽  
Antonio Alejandro Esperon ◽  
Ramon Ropero ◽  
Maria Caridad Rubio ◽  
Ronald Rodriguez ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Breast Cancer Patients ◽  
Brca1 And Brca2 ◽  
Brca2 Mutations ◽  
Brca1 And Brca2 Mutations
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