scholarly journals Molecular cloning of RhD cDNA derived from a gene present in RhD- positive, but not RhD-negative individuals

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 651-655 ◽  
Author(s):  
MA Arce ◽  
ES Thompson ◽  
S Wagner ◽  
KE Coyne ◽  
BA Ferdman ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Blood Group System ◽  
Pcr Cloning ◽  
Antigenic Protein ◽  
Genomic Libraries ◽  
Polymerase Chain ◽  
Rh Blood Group

The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.

scholarly journals Molecular cloning of RhD cDNA derived from a gene present in RhD- positive, but not RhD-negative individuals

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 651-655 ◽  
Author(s):  
MA Arce ◽  
ES Thompson ◽  
S Wagner ◽  
KE Coyne ◽  
BA Ferdman ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Blood Group System ◽  
Pcr Cloning ◽  
Antigenic Protein ◽  
Genomic Libraries ◽  
Polymerase Chain ◽  
Rh Blood Group

Abstract The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.

scholarly journals Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

2012 ◽  
Vol 49 (2) ◽  
pp. 67-70 ◽  
Author(s):  
M. Kolesárová ◽  
R. Herich ◽  
M. Levkut ◽  
J. Čurlík ◽  
M. Levkut
Keyword(s):  
Retrospective Studies ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Fresh Tissue ◽  
Carnoy’S Solution ◽  
Negative Results ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Cysticercus Ovis

AbstractPCR amplification of specific DNA regions is a powerful tool for retrospective studies, but not all preservation or fixation methods render DNA that is suitable for subsequent amplification. Several factors affect sensitivity of polymerase chain reaction (PCR) amplification. There were reported the effects of commonly used fixation solutions — 10 % neutral buffered formalin, 20 % neutral buffered formalin and Carnoy’s solution and the efficiency of PCR amplification in fresh tissue and paraffin (or wax) embedded samples of Cysticercus ovis. DNA from samples was isolated and PCR product of 1300 bp was amplified. Results indicated that the samples fixed in Carnoy’s solution produced reliable amplification of desired fragments. The samples that were fixed in 10 % and 20 % neutral buffered formalin brought negative results.

Characterization of nematode mitochondrial genomes.

2021 ◽  
pp. 250-264
Author(s):  
Danny A. Humphreys-Pereira ◽  
Taeho Kim ◽  
Joong-Ki Park
Keyword(s):  
Polymerase Chain Reaction ◽  
Mitochondrial Genome ◽  
Genome Annotation ◽  
Pcr Amplification ◽  
Gene Identification ◽  
Mitochondrial Genomes ◽  
Pcr Cloning ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.

scholarly journals Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2975-2980 ◽  
Author(s):  
S Simsek ◽  
BH Faas ◽  
PM Bleeker ◽  
MA Overbeeke ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
False Positive ◽  
Complete Agreement ◽  
Blood Group System ◽  
Chain Reaction ◽  
Group System ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Rh Blood Group

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3′ noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Simultaneous Phenotypically Distinct but Clonally Identical Mucosa-Associated Lymphoid Tissue and Follicular Lymphoma in a Patient With Sjögren’s Syndrome

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2247-2251 ◽  
Author(s):  
Antonella Aiello ◽  
Ming-Qing Du ◽  
Tim C. Diss ◽  
Huai-Zheng Peng ◽  
Francesco Pezzella ◽  
...  
Keyword(s):  
Lymph Node ◽  
Parotid Gland ◽  
Follicular Lymphoma ◽  
Malt Lymphoma ◽  
Lymphoid Tissue ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Chain Gene ◽  
Low Grade

A 44-year-old woman with a 12-year history of Sjögren’s syndrome (SS) developed a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in the parotid gland. Two years later, she presented with generalized lymphadenopathy and hepatosplenomegaly and a follicular lymphoma was diagnosed. To investigate the relationship of the two histologically distinct lymphomas, we re-examined their histology and immunophenotype and studied the lymphomatous tissue from the parotid, cervical lymph node, and spleen using molecular genetic methods. Histologic and immunophenotypic studies confirmed the previous diagnoses and also identified a previously unnoticed focus of follicular lymphoma in the second parotid gland biopsy. Polymerase chain reaction (PCR) amplification of the rearranged Ig heavy-chain gene showed the same sized dominant product in the MALT lymphoma and the follicular lymphoma. Similarly, PCR analysis of the t(14:18) translocation yielded an identical sized band from both MALT and follicular lymphoma. Cloning and sequencing of the Ig PCR products showed an identical CDR3 sequence from each lesion, indicating a common clonal lineage. The follicular lymphoma of the parotid gland lymph node and the follicular lymphoma of the spleen showed an identical mutation signature to that of the salivary gland MALT lymphoma. We propose that follicular lymphoma in the parotid gland lymph node may have resulted from colonization of lymphoid follicles by MALT lymphoma cells, following which the tumor cells were induced to express a follicular lymphoma phenotype, due to Bcl-2 overexpression caused by t(14;18), leading to a change in clinical behavior resulting in rapid widespread dissemination of disease. These observations suggest that the distinct phenotypes of low-grade B-cell lymphomas may be the consequence of interplay between genetic and local microenvironmental factors.

Cloning, characterization and expression analysis of a 1-aminocyclopropane-1-carboxylate synthase gene from pear

2013 ◽  
Vol 93 (3) ◽  
pp. 465-471
Author(s):  
Haiyan Shi ◽  
Yuxing Zhang ◽  
Liang Chen
Keyword(s):  
Gene Product ◽  
Expression Analysis ◽  
Cdna Clone ◽  
Phylogenetic Analyses ◽  
Pcr Amplification ◽  
Acc Synthase ◽  
Pcr Analysis ◽  
Pyrus Pyrifolia ◽  
Reading Frame ◽  
Amplification Technique

Shi, H., Zhang, Y. and Chen, L. 2013. Cloning, characterization and expression analysis of a 1-aminocyclopropane-1-carboxylate synthase gene from pear. Can. J. Plant Sci. 93: 465–471. In this study, a cDNA clone encoding putative 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) that catalyzes the conversion of S-adenosyl-L-methionine to ACC in ethylene biosynthetic pathway was isolated from a cDNA library produced using mRNA from pear (Pyrus pyrifolia). The cDNA clone, designated PpACS2, comprised an open reading frame of 1, 341 bp encoding a protein of 446 amino acids that shares high similarity with the known plant ACSs. Using PCR amplification technique, a genomic clone (GenBank accession number: KC146402) corresponding to PpACS2 was isolated and shown to contain two introns. The PpACS2 gene product shared 97% identity with an ACC synthase from pear (Pyrus communis). Phylogenetic analyses clearly placed the gene product in the ACC synthase cluster of plant ACS superfamily tree. Quantitative RT-PCR analysis indicated that the PpACS2 gene was preferentially expressed in young pear leaves and shoots. The transcript of PpACS2 gene was accumulated at relatively high levels in anthers, but no signal was detected in the petals and mesocarp of pear. These results suggest that the PpACS2 may participate in the regulation of ethylene production in pear leaves, shoots, and anthers.

Identification and localization of renal Na(+)-Ca2+ exchanger by polymerase chain reaction

1992 ◽  
Vol 263 (4) ◽  
pp. F680-F685 ◽  
Author(s):  
A. S. Yu ◽  
S. C. Hebert ◽  
S. L. Lee ◽  
B. M. Brenner ◽  
J. Lytton
Keyword(s):  
Polymerase Chain Reaction ◽  
Rat Kidney ◽  
Northern Analysis ◽  
Pcr Analysis ◽  
Kidney Cortex ◽  
Pcr Cloning ◽  
Chain Reaction ◽  
Cloning Strategy ◽  
Polymerase Chain ◽  
A Minor

The molecular identity of the renal Na(+)-Ca2+ exchanger was determined by a homology-based polymerase chain reaction (PCR) cloning strategy. Rat kidney RNA was amplified by PCR, using oligonucleotide primers based on regions of low degeneracy in the published canine cardiac Na(+)-Ca2+ exchanger cDNA sequence, and the products were subcloned and sequenced. A 452-bp clone (NCX1) was identified, which shares 89% nucleotide and 98% amino acid sequence identity with the canine cardiac exchanger, suggesting that they are products of the same gene. NCX1 was shown, by Northern analysis, to hybridize to an abundant major transcript of 7 kb and a minor one of approximately 14 kb both localized predominantly to kidney cortex. Microdissected tubule PCR analysis revealed that NCX1 was enriched in distal convoluted tubule compared with other cortical nephron segments. Such a location is consistent with a Na(+)-Ca2+ exchanger corresponding to NCX1 playing a major role in active Ca2+ reabsorption at this site.

scholarly journals Intraspecific Variations of Epimedium koreanum by Randomly and Specifically Amplified Polymorphic DNA Markers

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 454D-454
Author(s):  
Hak-Tae Lim
Keyword(s):  
Genetic Relationships ◽  
Pcr Amplification ◽  
Morphological Characters ◽  
Pcr Analysis ◽  
Specific Primers ◽  
Banding Patterns ◽  
Intraspecific Variations ◽  
Similarity Indices ◽  
Leaflet Number ◽  
Polymerase Chain

Randomly and specifically amplified polymorphic DNA banding patterns based on polymerase chain reaction (PCR) analysis were used to assess the intraspecific genetic variations and relationships within Epimedium koreanum populations. A collection of 21 individuals were classified as different accessions by morphological characters such as leaflet number, shape of leaf base, cauline length, plant height, and leaf area. PCR amplification using 12 primers out of 62 [60 random (10-mer) primers, one 15-mer primer (M13 core sequence), and (GGAT)4] resulted in 89 amplified DNA fragments with polymorphisms (80.9%) in all of the tested plants. Similarity indices between accessions were computed from PCR data, and genetic relationships among intraspecific variations were closely related at the levels ranging from 0.66 to 0.93. These DNA data were not matched well with those of morphological characters because they were divided into two major groups at the similarity coefficient value of 0.74. Primers (VII, VIII) gave rise to monomorphic bands in all of examined plants, but specific primers (M13 core and (GGAT)4 sequences) were found to be very valuable molecular markers to evaluate the interspecific variations in Epimedium koreanum.

scholarly journals The Genomes of the Non-Clearing-Zone-Forming and Natural-Rubber- Degrading Species Gordonia polyisoprenivorans and Gordonia westfalica Harbor Genes Expressing Lcp Activity in Streptomyces Strains

2008 ◽  
Vol 74 (8) ◽  
pp. 2288-2297 ◽  
Author(s):  
Daniel Bröker ◽  
David Dietz ◽  
Matthias Arenskötter ◽  
Alexander Steinbüchel
Keyword(s):  
Streptomyces Coelicolor ◽  
Pcr Amplification ◽  
Gel Permeation Chromatography ◽  
Amino Acid Identity ◽  
Pcr Analysis ◽  
Genomic Libraries ◽  
Reverse Transcription Pcr ◽  
Degrading Bacterium ◽  
Degrading Bacteria ◽  
Gordonia Polyisoprenivorans

ABSTRACT The latex-clearing protein (LcpK30) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp K30), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230- and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp VH2) and G. westfalica strain Kb1 (lcp Kb1) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to LcpK30. Recombinant lcp VH2 and lcp Kb1 harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp VH2 and lcp Kb1 encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp VH2 was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-LcpK30 immunoglobulin Gs, which were raised in this study, were rather specific for LcpK30 and did not cross-react with LcpVH2 and LcpKb1. A lcp VH2 disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp VH2 seems not to be essential for rubber degradation in G. polyisoprenivorans.

Sign in / Sign up

Export Citation Format

Share Document