A Single multiplexed allele-specific polymerase chain reaction for simultaneous detection of alpha1-antitrypsin S and Z mutations.

Alpha1-antitrypsin (AAT) deficiency is an inherited disorder that can cause lung disease in adults and liver disease in adults and children. The S and Z mutations are the two most common mutations found in the AAT-deficient patients. We have developed a simple multiplexed allele-specific-PCR to detect both the S and Z mutations and the corresponding wild-type alleles. Polymerase chain reaction (PCR) product could be resolved on an agarose gel or using any fluorescent gel detection system. We obtained accurate genotyping results for the four alleles; the S, Z, and their corresponding wild-type alleles for all investigated samples simultaneously. The approach described in this paper is rapid, cost effective, and reliable and can also be adaptable into any laboratory setting because of its simplicity.

Detection of ApoE E2, E3 and E4 alleles using MALDI-TOF mass spectrometry and the homogeneous mass-extend technology.

Apolipoprotein (Apo) E is one of the five main types of blood lipoproteins (A-E). It is synthesized primarily in the liver and brain and helps in transporting lipids from one place to another as well as facilitates the clearing of dietary fats, such as triglycerides, from the blood. The ApoE gene exists in three different forms: E2, E3 and E4. E3 is considered to be the normal form. Variants of the ApoE gene have been associated with various diseases. Developing an assay for the genotyping of ApoE variants for use both in clinical and large cohort based association settings would be extremely valuable and would require the use of a platform that has high-throughput capabilities and is highly accurate. Here we describe an assay for the simultaneous genotyping of the ApoE variants in a single bi-plex reaction and a single well using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the homogeneous mass-extend (hME) technology. The assay is robust, highly accurate and suitable for both clinical applications and for the genotyping of large disease cohorts. Moreover, the prevalence of ApoE variants in a cohort of Caucasians from the central Wisconsin area is outlined.