Detection of EGFR and BRAF mutations by competitive allele-specific TaqMan polymerase chain reaction in lung adenocarcinoma

Abstract The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

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

Blood ◽

10.1182/blood.v85.10.2975.bloodjournal85102975 ◽

1995 ◽

Vol 85 (10) ◽

pp. 2975-2980 ◽

Cited By ~ 48

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.

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Two Novel Nonradioactive Polymerase Chain Reaction-Based Assays of Dried Blood Spots, Genomic DNA, or Whole Cells for Fast, Reliable Detection of Z and S Mutations in the α1-Antitrypsin Gene

Clinical Chemistry ◽

10.1093/clinchem/38.10.2100 ◽

1992 ◽

Vol 38 (10) ◽

pp. 2100-2107 ◽

Cited By ~ 17

Author(s):

B S Andresen ◽

I Knudsen ◽

P K Jensen ◽

K Rasmussen ◽

N Gregersen

Keyword(s):

Polymerase Chain Reaction ◽

Genomic Dna ◽

Dried Blood Spots ◽

Chain Reaction ◽

Tissue Samples ◽

Whole Cells ◽

Blood Spots ◽

Allele Specific ◽

Polymerase Chain ◽

Alpha 1 Antitrypsin

Abstract Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are well suited for routine diagnosis.

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