scholarly journals Optimized conditions for cycle sequencing of PCR products.

1994 ◽  
Vol 3 (6) ◽  
pp. 359-360 ◽  
Author(s):  
J H Horton ◽  
M D Hagen ◽  
M S Ko
Keyword(s):  
Cycle Sequencing ◽  
Pcr Products ◽  
Optimized Conditions

scholarly journals Hepatitis B Transmission: Is Vertical Transmission the Major Route in Intermediate Endemic Areas? A Proof-of-Concept Study Based on Mother–Child Genotypes

2021 ◽  
Author(s):  
Ujjal Poddar ◽  
Mercilena Benjamin ◽  
Rakesh Aggarwal ◽  
Aditya Narayan Sarangi ◽  
Amrita Mathias ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Region Of Interest ◽  
Cycle Sequencing ◽  
Child Pair ◽  
Mother And Child ◽  
Pcr Products ◽  
Major Route ◽  
Genetic Sequences ◽  
Hepatitis B Transmission ◽  
Genotype A

The route of hepatitis B transmission is believed to be horizontal in India, though pediatric studies showed mother as source in the majority of chronic HBV (CHB) cases. We aimed at establishing the fact that mother–child transmission is the main route of acquisition by documenting genotypically identical viruses in mother–child pairs. Blood samples of consecutive children (≤18 years) with CHB and high DNA (>10,000 IU/mL) and their positive mother were collected from January 2013 to December 2015. Polymerase chain reaction (PCR) products of HBV-DNA were amplified and sequenced by using BigDye Terminator Cycle Sequencing Kit v3.1 and aligned with previously described sequences in the region of interest for genotypes A to G by using BioEdit software. Phylogenetic tree was generated using p-distance algorithm in MEGA software version 6. Genotyping of 59 (33 children and 26 mothers) subjects include genotype A in 24 (40.7%) and genotype D in 35 (59.3%). Both mother–child pair genotyping was possible in 25. The median age of 25 children (20 males) was 9 (interquartile range, IQR: 4–11). The distribution of genotypes among mother–child pairs was similar. The concordance between children and their mothers was 24 of 25 (96%). Evolutionary analyses showed significant similarities between mother and child sequences for both genotype A and D, suggesting thereby the same virus. In conclusion, mother–baby transmission seems to be the major route of acquisition of HBV in children in India and near-complete homology in genetic sequences between mother–child pairs is definite proof for that. However, a larger epidemiological study is required to substantiate our findings.

scholarly journals Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

2000 ◽  
Vol 38 (7) ◽  
pp. 2715-2721 ◽  
Author(s):  
George J. Hanna ◽  
Victoria A. Johnson ◽  
Daniel R. Kuritzkes ◽  
Douglas D. Richman ◽  
Javier Martinez-Picado ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Population Based ◽  
Cycle Sequencing ◽  
Immunodeficiency Virus ◽  
Pcr Products ◽  
Hybridization Sequencing

The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibitor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to high-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by at least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons. In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resistance-associated codon discordances, cycle sequencing also more commonly called a known resistance-associated amino acid than hybridization sequencing did. The nucleotide sequence in the vicinity of several codons with discordant calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of all 5,994 amino acids and 0 of 494 drug resistance-associated codons). At positions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement with the population cycle sequencing result. In summary, most RT codons were highly concordant by both methods of population-based sequencing, with discordances due in large part to genetic mixtures within or adjacent to discordant codons.

scholarly journals Optimized Conditions for Cloning PCR Products Into an Xcm I T-Vector

BioTechniques ◽  
1997 ◽  
Vol 22 (1) ◽  
pp. 40-44 ◽  
Author(s):  
Brian C. Schutte ◽  
Koustubh Ranade ◽  
Jonathan Pruessner ◽  
Nic Dracopoli
Keyword(s):  
Pcr Products ◽  
Optimized Conditions

scholarly journals Detection of ERG11 point mutations in Iranian fluconazoleresistant Candida albicans isolates

2019 ◽  
Author(s):  
Ali Sardari ◽  
Hossein Zarrinfar ◽  
Rasoul Mohammadi
Keyword(s):  
Candida Albicans ◽  
Fungal Infections ◽  
Point Mutations ◽  
Drug Binding ◽  
Molecular Techniques ◽  
Clinical Isolates ◽  
Vaginal Mucosa ◽  
Cycle Sequencing ◽  
Fluconazole Resistance ◽  
Pcr Products

Background and Purpose: Candidiasis is referred to a group of superficial anddeep-tissue fungal infections often caused by Candida albicans. The superficialinfections affect the oral, oropharynx, esophagus, and vaginal mucosa. The treatmentof choice for these infections is the use of azoles, such as fluconazole. However, theincreased use of these antifungal agents has led to the emergence of azole-resistantisolates of C. albicans. Different mechanisms have been suggested for thedevelopment of drug resistance, such as mutations in the encoding gene ERG11.Mutations in ERG11 result in changes in the ERG11p spatial construction and reducethe affinity between the protein and azole. This study aimed to determine thesusceptibility profile of C. albicans clinical isolates to fluconazole usingmicrodilution method. The present research was also targeted toward the detection ofmutations that might be related to fluconazole resistance by the amplification andsequencing of ERG11 gene.Materials and Methods: This study was conducted on a total of 216 clinical isolatesobtained from Mashhad, Isfahan, and Tehran cities in Iran, during 2016-2018. Theclinical isolates were identified using molecular techniques. Furthermore, minimuminhibitory concentration (MICs) was determined according to the clinical and laboratorystandards institute M27-A3 and M27-S4 documents. The concentration range forfluconazole was obtained as 0.063-64 μg/ml. In the resistant strains, ERG11 genes wereamplified by specific primers. Subsequently, cycle sequencing reactions were performedon purified polymerase chain reaction (PCR) products in forward and reverse directions.Finally, the results were analyzed by MEGA (version 7) and Gene Runner software(version 6.5.30).Results: Out of 216 strains, 100 (46.3%) species were identified as C. albicans. TheMIC values for fluconazole had a range of 0.125-16 μg/ml with the MIC50 and MIC90values of 0.5 and 1 μg/ml, respectively. Totally, 41 nucleotide changes were detectedamong 4 resistant isolates. In this regard, 4 out of 41 mutations in codons caused changesin ERG11p; however, these mutations did not lead to fluconazole resistance.Conclusion: Fluconazole resistance among clinical isolates is not merely due to thechanges in ERG11p. This resistance may be also related to some other mechanisms, suchas the prevention of the intracellular accumulation of the antifungal agent and alterationof the target enzyme to diminish drug binding.

SDHA and SDHB mutations in KIT/PDGFRA WT gastrointestinal stromal tumors.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10087-10087 ◽  
Author(s):  
Margherita Nannini ◽  
Maria A. Pantaleo ◽  
Annalisa Astolfi ◽  
Milena Urbini ◽  
Serena Formica ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Massively Parallel Sequencing ◽  
Heterozygous Mutation ◽  
Missense Mutations ◽  
Wild Type ◽  
Cycle Sequencing ◽  
Sequencing Method ◽  
Pcr Products ◽  
Sdhb Gene ◽  
Genetic Analyzer

10087 Background: KIT/PDGFRA wild-type (WT) GISTs harbour mutations on SDHB and SDHC and, more recently, we described mutations on SDHA using massively parallel sequencing approach. We sequenced SDHA and SDHB genes in a larger series in order to validate the data. Methods: SDHA gene (1-15 exons) and SDHB gene (1-8 exons) (even not all exons in all samples) were sequenced on tumor (T) and/or peripheral blood (PB) of WT GIST patients by Sanger Sequencing method. DNA was extracted from tumor specimens by the QIAmp DNA Mini kit (Qiagen, Milan, Italy) and amplified with specific primer pairs designed to amplify exons but not SDHA pseudo-genes located on chromosomes 3 and 5. Then, PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Milan, Italy) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). Sanger sequencing was performed on ABI 3730 Genetic Analyzer (Applied Biosystems). Results: SDHA gene exons were sequenced on a total of 27 WT GIST patients, in particular on T, PB and both from 12, 6 and 9 patients respectively. SDHB gene exons were sequenced on a total of 18 out of 27 patients, in particular on T, PB and both from 7, 8 and 3 patients respectively. 8 SDHA mutations were found in 5 samples (18.5%). Besides those previously identified, 5 new SDHA mutations were found in other 3 samples: one sample harboured R171C and R589Q heterozygous missense mutation in exons 5 and 13 respectively. The other one harboured G419R and E564K heterozygous missense mutations in exons 9 and 13 respectively. The third sample harboured a delCAG immediately upstream of exon 5, in heterozygosis on PB and in homozygosis on T. A SDHB heterozygous mutation (301delT) in exon 4 was found on 1 PB sample. Conclusions: the presence of SDHA mutations has been confirmed in a subgroup of WT GIST patients. All subunits of SDH complex should be sequenced on WT GIST patients in order to explore the frequency and any linkage between each other and the pathogenetic and clinical significance.

scholarly journals Identification of Tinomiscium petiolare from Vietnam using the DNA barcode

2021 ◽  
Vol 182 (2) ◽  
pp. 114-122
Author(s):  
B. B. Thinh ◽  
R. V. Doudkin ◽  
L. D. Chac ◽  
H. V. Chinh ◽  
Q. V. Hoi ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Medicinal Species ◽  
Cycle Sequencing ◽  
Stage Of Development ◽  
Pcr Products ◽  
Genetic Sequences ◽  
Total Dna

Background. Tinomiscium petiolare Hook.f. & Thomson is a medicinal species of the family Menispermaceae. This species is currently being intensively exploited for therapeutic purposes. Precise and rapid identification of T. petiolare is critical and essential for the classification, propagation, use and conservation of its genetic resources. In recent years, DNA barcoding has been known to be a fast and sensitive method for identifying species at any stage of development, using short DNA sequences. In this study we have performed the identification of T. petiolare specimens in Vietnam based on the sequence analysis of 4 DNA barcode loci: ITS, matK, rbcL and rpoC.Materials and methods. Total DNA was extracted from leaf samples using DNeasy Plant Mini Kit. PCR amplification of the ITS, matK, rbcL and rpoC regions was carried out on the GeneAmp PCR System 9700 with specific primers. The purified PCR products were sequenced on the ABI 3500 Genetic Analyzer system, using BigDye®Terminator v3.1 Cycle Sequencing Kit. These genetic sequences were analyzed and compared, and a phylogenetic tree was constructed using BioEdit, BLAST, and MEGA 6 programs.Results and conclusion. The success rate of amplification and sequencing was 100% for all 4 DNA barcode loci (ITS, matK, rbcL and rpoC) in the studied specimens. The produced sequence sizes of ITS, matK, rbcL and rpoC in the specimens were 574 bp, 810 bp, 527 bp and 488 bp, respectively. Further, we identified that all studied specimens were genetically related to each other and associated with the same species T. petiolare. Overall, the results of the study generated the most complete DNA barcode database of T. petiolare collected in Vietnam, contributing to the taxonomy and identification of this species. 

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