scholarly journals Rh E/e genotyping by allele-specific primer amplification

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 829-832 ◽  
Author(s):  
BH Faas ◽  
S Simsek ◽  
PM Bleeker ◽  
MA Overbeeke ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Nucleotide Position ◽  
Blood Group Antigens ◽  
Specific Primer ◽  
Nucleotide Difference ◽  
Homologous Genes ◽  
Allele Specific ◽  
Antisense Primer ◽  
Polymerase Chain ◽  
Sense Primer ◽  
Rh Blood Group

It has been shown that the Rhesus (Rh) blood group antigens are encoded by two homologous genes: the Rh D gene and the Rh CcEe gene. The Rh CcEe gene encodes different peptides: the Rh C, c, E, and e polypeptides. Only one nucleotide difference has been found between the alleles encoding the Rh E and the Rh e antigen polypeptides. It is a C-- >G transition at nucleotide position 676, which leads to an amino acid substitution from proline to alanine in the Rh e-carrying polypeptide. Here we present an allele-specific primer amplification (ASPA) method to determine the Rh E and Rh e genotypes. In one polymerase chain reaction, the sense primer had a 3′-end nucleotide specific for the cytosine at position 676 of the Rh E allele. In another reaction, a sense primer was used with a 3′-end nucleotide specific for the guanine at position 676 of the Rh e allele and the Rh D gene, whereas the antisense primer had a 3′-end nucleotide specific for the adenine at position 787 of the Rh CcEe gene. We tested DNA samples from 158 normal donors (including non-Caucasian donors and donors with rare Rh phenotypes) in these assays. There was full concordance with the results of serologic Rh E/e phenotyping. Thus, we may conclude that the ASPA approach leads to a simple and reliable method to determine the Rh E/e genotype. This can be useful in Rh E/e genotyping of fetuses and/or in cases in which no red blood cells are available for serotyping. Moreover, our results confirm the proposed association between the cytosine/guanine polymorphism at position 676 and the Rh E/e phenotype.

scholarly journals Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1196-1201
Author(s):  
I Mouro ◽  
Y Colin ◽  
P Sistonen ◽  
PY Le Pennec ◽  
JP Cartron ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
Point Mutations ◽  
Blood Group Antigens ◽  
Chain Reaction ◽  
Definitive Evidence ◽  
Allele Specific ◽  
The Common ◽  
Polymerase Chain ◽  
Rh Blood Group

The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

scholarly journals Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1196-1201 ◽  
Author(s):  
I Mouro ◽  
Y Colin ◽  
P Sistonen ◽  
PY Le Pennec ◽  
JP Cartron ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
Point Mutations ◽  
Blood Group Antigens ◽  
Chain Reaction ◽  
Definitive Evidence ◽  
Allele Specific ◽  
The Common ◽  
Polymerase Chain ◽  
Rh Blood Group

Abstract The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

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.

RHD gene deletion occurred in the Rhesus box

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3662-3668 ◽  
Author(s):  
Franz F. Wagner ◽  
Willy A. Flegel
Keyword(s):  
Open Reading Frames ◽  
Chromosome 1 ◽  
Nucleotide Sequencing ◽  
Blood Group Antigens ◽  
Specific Detection ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Specific Priming ◽  
Rh Blood Group ◽  
Reading Frames

Abstract The Rh blood group antigens derive from 2 genes,RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a deletion of the whole RHD gene occurs with a frequency of approximately 40%. The relative position of the 2 RH genes and the location of the RHD deletion was previously unknown. A model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and nucleotide sequencing. The open reading frames of bothRH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30 000 base pair (bp) that contained the SMP1 gene. The RHD gene was flanked by 2 DNA segments, dubbed Rhesus boxes, with a length of approximately 9000 bp, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1463 bp of identity. PCR with sequence-specific priming (PCR-SSP) and PCR with restriction fragment length polymorphism (PCR-RFLP) were used for specific detection of the RHDdeletion. The molecular structure of the RH gene locus explains the mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of theRHD− genotype is now possible.

scholarly journals The Efficacy of Molecular Markers Analysis with Integration of Sensory Methods in Detection of Aroma in Rice

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
H. Y. Yeap ◽  
G. Faruq ◽  
H. P. Zakaria ◽  
J. A. Harikrishna
Keyword(s):  
Sensory Evaluation ◽  
Molecular Analysis ◽  
Aromatic Rice ◽  
Phenotypic Analysis ◽  
Genotypic Analysis ◽  
Allele Specific ◽  
Antisense Primer ◽  
Specific Amplification ◽  
Rice Genotypes ◽  
Sense Primer

Allele Specific Amplification with four primers (External Antisense Primer, External Sense Primer, Internal Nonfragrant Sense Primer, and Internal Fragrant Antisense Primer) and sensory evaluation with leaves and grains were executed to identify aromatic rice genotypes and their F1individuals derived from different crosses of 2 Malaysian varieties with 4 popular land races and 3 advance lines. Homozygous aromatic (fgr/fgr) F1individuals demonstrated better aroma scores compared to both heterozygous nonaromatic (FGR/fgr) and homozygous nonaromatic (FGR/FGR) individuals, while, some F1individuals expressed aroma in both leaf and grain aromatic tests without possessing thefgrallele. Genotypic analysis of F1individuals for thefgrgene represented homozygous aromatic, heterozygous nonaromatic and homozygous nonaromatic genotypes in the ratio 20 : 19 : 3. Genotypic and phenotypic analysis revealed that aroma in F1individuals was successfully inherited from the parents, but either molecular analysis or sensory evaluation alone could not determine aromatic condition completely. The integration of molecular analysis with sensory methods was observed as rapid and reliable for the screening of aromatic genotypes because molecular analysis could distinguish aromatic homozygous, nonaromatic homozygous and nonaromatic heterozygous individuals, whilst the sensory method facilitated the evaluation of aroma emitted from leaf and grain during flowering to maturity stages.

Rapid diagnosis of familial defective apolipoprotein B-100 by Amplification Refractory Mutation System

1991 ◽  
Vol 37 (11) ◽  
pp. 1983-1987 ◽  
Author(s):  
P R Wenham ◽  
C R Newton ◽  
R S Houlston ◽  
W H Price
Keyword(s):  
Apolipoprotein B ◽  
Clinical Chemistry ◽  
Amplification Refractory Mutation System ◽  
Specific Primer ◽  
Chain Reaction ◽  
Apo B ◽  
Mutation System ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Template Dna

Abstract We report a method for the diagnosis of familial defective apolipoprotein (apo) B-100, using the Amplification Refractory Mutation System (ARMS) and either whole blood or extracted DNA in the polymerase chain reaction. Normal and mutant alleles are identified by using two allele-specific oligonucleotide primers, each with the same common primer, to amplify a 187-bp fragment of the apo B-100 gene. Fragment amplification occurs only when the allele-specific primer matches the nucleotide sequence of the template DNA. The amplification product is detected by agarose gel electrophoresis, followed by staining with ethidium bromide. The technique is simple, reliable, and robust. It avoids the use of radiation or hybridization with allele-specific oligonucleotide probes, and is well suited for use in the routine clinical chemistry department.

RHD gene deletion occurred in the Rhesus box

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3662-3668 ◽  
Author(s):  
Franz F. Wagner ◽  
Willy A. Flegel
Keyword(s):  
Open Reading Frames ◽  
Chromosome 1 ◽  
Nucleotide Sequencing ◽  
Blood Group Antigens ◽  
Specific Detection ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Specific Priming ◽  
Rh Blood Group ◽  
Reading Frames

The Rh blood group antigens derive from 2 genes,RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a deletion of the whole RHD gene occurs with a frequency of approximately 40%. The relative position of the 2 RH genes and the location of the RHD deletion was previously unknown. A model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and nucleotide sequencing. The open reading frames of bothRH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30 000 base pair (bp) that contained the SMP1 gene. The RHD gene was flanked by 2 DNA segments, dubbed Rhesus boxes, with a length of approximately 9000 bp, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1463 bp of identity. PCR with sequence-specific priming (PCR-SSP) and PCR with restriction fragment length polymorphism (PCR-RFLP) were used for specific detection of the RHDdeletion. The molecular structure of the RH gene locus explains the mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of theRHD− genotype is now possible.

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