scholarly journals Rapid Screening of Plasmid DNA by Direct Sequencing from Bacterial Colonies

BioTechniques ◽  
2000 ◽  
Vol 29 (3) ◽  
pp. 436-438 ◽  
Author(s):  
Jennifer G. Brown Gladden ◽  
Reena Ray ◽  
R. Daniel Gietz ◽  
Michael R.A. Mowat
Keyword(s):  
Plasmid Dna ◽  
Direct Sequencing ◽  
Rapid Screening

scholarly journals High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC

2001 ◽  
Vol 47 (3) ◽  
pp. 548-555 ◽  
Author(s):  
Elke Schaeffeler ◽  
Thomas Lang ◽  
Ulrich M Zanger ◽  
Michel Eichelbaum ◽  
Matthias Schwab
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
False Negative ◽  
Rapid Screening ◽  
Tpmt Activity ◽  
Polymorphism Analysis ◽  
Specific Pcr ◽  
Restriction Fragment Polymorphism ◽  
Allele Specific ◽  
Tpmt Gene

Abstract Background: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC). Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR). Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals. Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.

scholarly journals A rapid screening with direct sequencing from blood samples for the diagnosis of Leigh syndrome

2014 ◽  
Vol 1 ◽  
pp. 133-138 ◽  
Author(s):  
Hiroko Shimbo ◽  
Mariko Takagi ◽  
Mitsuko Okuda ◽  
Yu Tsuyusaki ◽  
Kyoko Takano ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Leigh Syndrome ◽  
Rapid Screening ◽  
Blood Samples

Rapid screening of SNPs in metastatic colorectal cancer (mCRC) utilizing multiplex sequencing technology (Sequenom).

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 418-418
Author(s):  
Radhashree Maitra ◽  
Jay B. Nayak ◽  
Atrayee Basu-Mallick ◽  
Arjun Sood ◽  
Titto A Augustine ◽  
...  
Keyword(s):  
High Throughput ◽  
Direct Sequencing ◽  
Cost Effective ◽  
Rapid Screening ◽  
Accurate Identification ◽  
Multiple Snps ◽  
Fast Screening ◽  
Effective Manner ◽  
Multiplex Sequencing ◽  
Multiple Samples

418 Background: Accurate and fast screening of mutations is essential for designing individualized therapy necessary and critical for efficient disease management and better patient outcome in mCRC. Detection of hotspots by gold standard direct sequencing (DS) is time consuming and cost ineffective. Pyrosequencing (PS) technique is rapid and precisely committed towards SNP detection. Recent introduction of high throughput multiplex PCR based extension on microarray (Sequenom, SEQ) offers a robust platform capable of detecting multiple SNPs simultaneously in a rapid and cost effective manner. The current study analyzes the concordance and efficacy of the cutting edge SEQ technique to the well established DS and PS methods. Methods: DNA isolated from 122 specimens from 76 mCRC patients were sequenced by all three methods. DS and PS were performed on 4 genes at 10 hotspots. SEQ multiplexing was performed on 31 hotspots in 19 genes by 4 multiplex reactions. Results: We were able to make "calls" for all samples by DS and PS. With the multiplex system, the “calls” rate was 97.8% of successful reactions. Using PS data as our standard in the assay we calculated the percent concordance of DS and SEQ. Futhermore SEQ offered a more accurate identification of the substituted nucleotide in Kras codon 12 as compared to PS. Conclusions: The multiplexing of PCR reactions offers an excellent advantage of high throughput with strong feasibility of analyzing several samples for multiple SNPs simultaneously. The concordance rate of > 90% when compared to PS along with the ability to analyze multiple samples/ hotspots plexed together in a time effective rapid mode provided a trifold advantage of the sequenom technology. It is therefore the next generation technology for rapid genetic evaluation of cancer patients. [Table: see text]

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4261-4261
Author(s):  
Gonzalo Manrique ◽  
Roberta Bittencout ◽  
Verónica Pérez ◽  
Vanesa Sholl ◽  
Monica Cappetta ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Low Cost ◽  
Mutation Screening ◽  
Point Mutations ◽  
Kinase Domain ◽  
Rapid Screening ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Allele Specific

Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.

scholarly journals Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae

1999 ◽  
Vol 43 (2) ◽  
pp. 297-301 ◽  
Author(s):  
Danièle Choury ◽  
Gérald Aubert ◽  
Marie-France Szajnert ◽  
Kemal Azibi ◽  
Marc Delpech ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Vibrio Cholerae ◽  
Plasmid Dna ◽  
Direct Sequencing ◽  
Clinical Strain ◽  
Acid Protein ◽  
K 12 ◽  
Amino Acid Protein

ABSTRACT A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new β-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB β-lactamases and to SAR-1, another carbenicillin-hydrolyzing β-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This β-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of theStaphylococcus aureus PC-1 β-lactamase.

scholarly journals A Novel Variant in Iranian Patient with Cystinuria: A Case Report

2021 ◽  
Author(s):  
Ali Mardi ◽  
Hamed Heidary ◽  
Seyyed Mohammad Mousavi ◽  
Ghasem Khazaei ◽  
Eskandar Taghizadeh
Keyword(s):  
Sequence Analysis ◽  
Direct Sequencing ◽  
Autosomal Recessive Disorder ◽  
Protein Product ◽  
Rapid Screening ◽  
Iranian Patient ◽  
Gene Sequence Analysis ◽  
Novel Variant ◽  
Slc3a1 Gene ◽  
East Azerbaijan

Cystinuria is an autosomal recessive disorder in which the renal reabsorption of cystine, arginine, lysine and ornithine are disturbed. The two genes, the pathogenic forms of which are responsible for the disorder, are SLC7A9 and SLC3A1. In this study, we describe a disease that has a new c.916A> T variant (p. K306 *) in exon 5 of the SLC3A1 gene. This variant results in the NMD phenomenon in which the protein product is not produced because of mRNA destruction. In 2020, blood sample of a 41-yr-old man from east Azerbaijan, Iran together with his parents were collected to be studied. PCR and direct sequencing were performed to detect the possible SLC3A1 variant. Whole-gene sequence analysis done by Mutation surveyor Software revealed a novel nonsense homozygous variant in exon 5 of the gene. Parental Sequence Analysis shows that they are heterozygous. According to ACMG guideline, this variant is considered as pathogen. Finding serious mutations can allow rapid screening for cystinuria by analyzing common mutations. It should also be considered as a pathogenic variant in patients’ cystinuria.

scholarly journals “Colony Sequencing”: Direct Sequencing of Plasmid DNA from Bacterial Colonies

BioTechniques ◽  
1997 ◽  
Vol 22 (3) ◽  
pp. 412-418 ◽  
Author(s):  
Christian Kilger ◽  
Matthias Krings ◽  
Hendrik Poinar ◽  
Svante Pääbo
Keyword(s):  
Plasmid Dna ◽  
Direct Sequencing

Detection of paralogous polyketide synthase genes in Parmeliaceae by specific primers

2005 ◽  
Vol 38 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Magdalena OPANOWICZ ◽  
Juliane BLAHA ◽  
Martin GRUBE
Keyword(s):  
Polyketide Synthase ◽  
Direct Sequencing ◽  
Polyketide Synthases ◽  
Rapid Screening ◽  
Specific Primers ◽  
Spliceosomal Introns ◽  
The Family ◽  
Pcr Products ◽  
Signature Sequences ◽  
Selection Of

A first assessment of paralogy in non-reducing polyketide synthases of Parmeliaceae is presented. Primers which are specific to the keto-acyl synthase domain were used to amplify gene fragments of putative non-reducing polyketide synthases from various representatives of the family. The corresponding sequences were analysed together with a selection of known polyketide synthase genes from other fungi, including lichenized fungi. The results suggest that genes from Parmeliaceae represent at least 6 paralogs. Their different positions in the tree partly correlate with the variable presence of spliceosomal introns at particular positions in the gene fragments. Because only one paralog could be unambiguously detected in each species by direct sequencing of PCR products with this approach, we tested the applicability of clade-specific primers, designed by using orthologous signature sequences. With these primers more paralogs could be detected from the same DNA extract in a number of species, but certain paralogs were consistently not amplified in these species. The paralog-specific primer approach can potentially be used for a rapid screening of PKS genes from a broader range of lichen fungi.

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