Pseudogene of dihydrolipoyl succinyltransferase (E2k) found by PCR amplification and direct sequencing of rodent-human cell hybrid DNAs

1994 ◽  
Vol 20 (4) ◽  
pp. 339-343 ◽  
Author(s):  
Xingang Cai ◽  
Paul Szabo ◽  
Ghazala Ali ◽  
Rudolph E. Tanzi ◽  
John P. Blass
Keyword(s):  
Human Cell ◽  
Cell Hybrid ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Hybrid Dnas

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

scholarly journals Disseminated mycobacteriosis caused by Mycobacterium kansasii in a pot-bellied pig

2018 ◽  
Vol 30 (4) ◽  
pp. 646-650 ◽  
Author(s):  
Ryan Schafbuch ◽  
Stacy Tinkler ◽  
Chee Kin Lim ◽  
Rebecca Wolking ◽  
José Ramos-Vara
Keyword(s):  
Direct Sequencing ◽  
Pcr Amplification ◽  
Etiologic Agent ◽  
Type Ii ◽  
Mycobacterium Kansasii ◽  
Mycobacterial Species ◽  
Computed Tomography Scan ◽  
History Of ◽  
Tomography Scan

A 1.5-y-old spayed female Juliana pot-bellied pig was presented to the Purdue University Veterinary Teaching Hospital with a history of wasting and anorexia. Enlarged and partially mineralized lymph nodes were identified on radiographs and computed tomography scan. Generalized lymphadenomegaly and disseminated nodules in the lungs, liver, spleen, and kidneys were identified on postmortem examination. Histologic examination revealed caseonecrotic granulomas with numerous intracellular, acid-fast bacilli. Mycobacterium kansasii type II was identified as the etiologic agent by PCR amplification using universal Mycobacterium primers, direct sequencing of the PCR amplicon, and comparison to sequences in GenBank. We describe a case in a pot-bellied pig of mycobacteriosis caused by an atypical mycobacterial species and highlight the important role of laboratory testing in suspected cases of tuberculosis.

scholarly journals A myeloid-related sequence that localizes to human chromosome 8q21.1-22

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1713-1719 ◽  
Author(s):  
WM Mars ◽  
P van Tuinen ◽  
HA Drabkin ◽  
JW White ◽  
GF Saunders
Keyword(s):  
Amino Acid ◽  
Human Chromosome ◽  
Somatic Cell ◽  
Southern Blot ◽  
Cell Hybrid ◽  
Somatic Cell Hybrid ◽  
Chromosome 8 ◽  
Nucleotide Sequence Analysis ◽  
Related Sequence ◽  
Hybrid Dnas

Abstract A myeloid-related sequence (mrs) has previously been identified that is highly expressed in selected subpopulations of myeloid leukocytes. Nucleotide sequence analysis indicates that mrs encodes what is apparently a unique 93-amino acid protein that includes an 18-amino acid leader sequence. Hybridization of an mrs cDNA probe to a Southern blot made from somatic cell hybrid DNAs shows 100% concordance with human chromosome 8, thus indicating that mrs localizes to this chromosome. In situ hybridization to metaphase chromosomes further sublocalizes mrs to bands 8q21.1–23 as 58% of the grains displayed on chromosome 8 were clustered in this region. This area encompasses the translocation breakpoint 8q22, which is rearranged in an estimated 18% of patients diagnosed with the M2 subclassification of acute nonlymphocytic leukemia (M2-ANLL). When Southern blot hybridization was performed by using somatic cell hybrid DNAs harboring either a single 8q- or a single 21q+ chromosome from two different patients with M2- ANLL, a signal was only detected in the hybrid containing the 8q- chromosome.

scholarly journals p53 Protein Detected By Immunohistochemical Staining is Not Always Mutant

1993 ◽  
Vol 11 (5-6) ◽  
pp. 239-250 ◽  
Author(s):  
Catriona Macgeoch ◽  
Diana M. Barnes ◽  
Julia A. Newton ◽  
Shehla Mohammed ◽  
Shirley V. Hodgson ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Direct Sequencing ◽  
P53 Protein ◽  
Pcr Amplification ◽  
P53 Gene ◽  
Tumour Suppressor Gene ◽  
Nuclear Staining ◽  
Breast Carcinomas ◽  
Direct Dna Sequencing ◽  
P53 Antibodies

The expression of the tumour suppressor gene p53 was analyzed in a variety of human solid tumours by immunohistochemistry and direct DNA sequencing. Positive nuclear staining using a panel of anti-p53 antibodies was used to select tumours for further genetic analysis. Using PCR amplification followed by immobilization onto magnetic beads and direct sequencing, we sequenced exons 5-9 of the p53 gene fro m 9 melanomas, 8 nasopharyngeal carcinomas, 16 sporadic breast carcinomas and 11 patients from familial breast cancer families. No sequence alterations of the p53 gene were detected in either the melanoma or nasopharyngeal tumours and only 19% of the primary breast carcinomas showed a variant band indicative of a mutation. Our results indicate firstly that p53 mutations are not generally involved in the tumour types studied and secondly the data emphasize the disparity encountered when attempting to correlate p53 immunohistochemical positivity with mutations within the p53 gene.

scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

scholarly journals Association of Lumican Gene with Susceptibility to Pathological Myopia in the Northern Han Ethnic Chinese

2009 ◽  
Vol 2009 ◽  
pp. 1-5 ◽  
Author(s):  
Fengju Zhang ◽  
Tingzhun Zhu ◽  
Zhongjun Zhou ◽  
Yudong Wu ◽  
Yang Li
Keyword(s):  
Direct Sequencing ◽  
Univariate Analysis ◽  
Regulatory Region ◽  
Pcr Amplification ◽  
Han Chinese ◽  
Genotype Distribution ◽  
Pathological Myopia ◽  
Sporadic Group ◽  
Ethnic Chinese ◽  
Significant Difference

Pathological myopia is a severe hereditary ocular disease leading to blindness. It is urgent and very important to find the pathogenesis and therapy for this disease. The purpose of the study is to analyze sequences of lumican and decorin genes with pathological myopia(PM) and control subjects to verify the relationship between lumican, decorin genes and PM in Northern Han Chinese. We collected and analyzed the blood samples of 94 adults (including 12 pedigree cases and 82 sporadic cases) with PM and 90 controls in the northern Han ethnic Chinese. Genotyping was performed by direct sequencing after polymerase chain reaction(PCR) amplification and allele frequencies were tested for Hardy-Weinberg equilibrium. Univariate analysis revealed significant differences between two groups for three SNPs: rs3759223(C→T)and rs17853500(T→C)of the lumican gene and rs74419(T→C)of decorin gene with (P<.05) for all their genotype distribution and allele frequency. There is no significant difference for incidence of these mutations between pedigree and sporadic group (P>.05). The results suggested that the sequence variants in 5′-regulatory region of lumican gene and 3'UTR of decorin gene were associated significantly with PM in Northern Han Chinese. Further studies are needed to confirm finally whether the two genes are the virulence genes of PM.

Analysis of Immunogenetic Factors in Paroxysmal Nocturnal Hemoglobinuria Reveal Increased Frequency of IL-10 Low Secretor Genotype and Correlates with Induced IL-10 Production.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 983-983
Author(s):  
Giridharan Ramsingh ◽  
Zachary Nearman ◽  
Chris Hung ◽  
Serio Bianca ◽  
Antonio Risitano ◽  
...  
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Hla Typing ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Hla Ligand ◽  
Functional Consequences ◽  
Immunologic Diseases ◽  
Common Pathogenesis

Abstract Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) have a common pathogenesis related to an immune attack on hematopoietic progenitor and stem cells. While the inciting events are not known, it is possible that complex immunogenetic predisposition factors exist which, in the context of exogenous influences, determine risk for these diseases. They include HLA background, KIR genotype, cytokine and immunomodulatory gene polymorphisms as well as gene variants involved in peptide processing and presentation. We have empirically selected a number of important polymorphisms that were described in the context of various immunologic diseases and studied their frequency in a large cohort (N=57) of PNH patients. As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of Nk-cells and cytotoxic T lymphocytes, we examined the combined impact of KIR/KIR-L genotypes on the risk of PNH and PNH/AA syndrome. PNH showed a decreased frequency of inhibitory KIR-2DL1 and KIR-2DL3 genes (79% vs. 95%, p=.0054; 67% vs. 89%, p=.0032). Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, deduced from HLA typing, revealed an increased frequency of unopposed 2DS2 (2DS2/C1 type, 37% vs.10%, p=.012) and 2DL2 (2DL2/C1 mismatch 37% vs 11%, p=.031) but these mismatches have a potentially opposing functional effect. Using sequence-specific PCR amplification and/or direct sequencing, we have genotyped DNA samples derived from our cohort who presented with different subtypes of PNH and studied single nucleotide polymorphisms (SNPs) in cytokine genes such as TNF-a (−308 G/A), TGF-b 1 (C/T codon 10, C/G codon 25), IL-10 (−1082 G/A), and IFN-g (+874 A/T) and immunomodulatory receptor genes like CTLA-4 exon 6 (+49 G/A), FcγRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). These SNPs are responsible for intrinsic differences in cytokine production and receptor function and can thereby influence immune physiologic and pathologic responses. PNH patients showed a significantly higher frequency of A/A polymorphisms at intron −1082 of the IL-10 gene consistent with a “low secretor phenotype” (36% PNH (N=50) vs 13.8% controls (N=363), p=0.0001). In contrast to a few smaller studies, no association with any other SNP tested was found. To establish whether this finding translates into functional consequences, we examined basal and PMA-induced IL-10 secretion in PNH patients andcontrols. Upon induction, IL-10 production increased in controls (N=5, 14.11± 9.8), while in general, stimulation resulted in a much weaker IL-10 response in PNH patients (N=6, 4.51± 2.16). However, when compared genotypic IL-10 “low secretors” (−1082 A/A) among PNH patients showed significantly lower induction levels of IL-10 (N=2, 3.1± 1.58) as compared to PNH patients with “normal/high secretor” genotypes (−1082 A/G,G/G) ((N=4, 5.22 +/−3.1). The low secretor IL-10 genotype may correspond to the exaggerated TH1 response in PNH and in general supports the notion that complex inherited traits may exist that genetically determine propensity to PNH evolution.

Validation study of direct sequencing and PCR-invader for detecting EGFR mutations in non-small cell lung cancer (NSCLC)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8094-8094
Author(s):  
Y. Naito ◽  
K. Goto ◽  
H. Kenmotsu ◽  
Y. Nishiwaki ◽  
K. Kubota ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
The Other ◽  
Egfr Mutations ◽  
Wild Type ◽  
Highly Sensitive ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 19 Deletion ◽  
Exon 19

8094 Background: EGFR mutation is both a predictive and prognostic factor for NSCLC treated with EGFR-TKI. Although new, highly sensitive methods for detecting EGFR mutations are currently available, these methods have not been validated. Methods: To validate direct sequencing and PCR-invader for detecting EGFR mutation, we analyzed 124 NSCLC by both methods concomitantly. Tumor tissues were obtained by surgical resection. Formalin-fixed paraffin-embedded specimens were prepared to analyze EGFR mutation. In direct sequencing, Exon 18, 19, and 21 of the EGFR gene were amplified, and PCR amplification products were sequenced directly (Mitsubishi Chemical Medience Corporation). PCR-invader was performed using the invasive cleavage of probe oligonucleotides to detect 10 mutations including Exon 18, 19, 20, 21 (BML incorporation). Results: EGFR mutations were detected in 51 patients (41%) by direct sequencing and 56 (45%) by PCR-invader. Discordance between two methods was found in 12 patients (10%). Exon 19 deletion was detected in 18 and 22 patients respectively. Exon 21 L858R was detected in 30 and 32 patients respectively. Each mutation in exon 19 deletions or L858R detected by direct sequencing could also be identified by PCR-invader. Overall 45 mutations were concordant by both methods. In two patients who received gefitinib, one patient with wild type by both methods did not respond to gefitinib. On the other hand, the other patient expressing Exon 19 deletion by PCR-invader but regarded as wild type by direct sequencing responded to gefitinib monotherapy. Conclusions: Discrepancy between two methods for detecting EGFR mutation was demonstrated and PCR-invader seems to be more sensitive. Further investigation including other highly sensitive methods is currently underway. No significant financial relationships to disclose.

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