Science Journal of Biochemistry
October 2013, Volume 2013, ISSN: 2276-6294
© Author(s) 2013. This work is distributed under the Creative Commons Attribution 3.0 License.
A Single-step Diagnostic Lightcycler Assay for Detection of Lactose Tolerance Gene Variants In the MCM6 -13907 to -13915 Region
Matthias Ballhause*†1, Merete Rasmussen†2, Thomas V.F. Hviid2, Jesper T. Troelsen3, Lars Ødum2 and Olfert Landt1
1TIB MOLBIOL Syntheselabor GmbH, Eresburgstraße 22-23, D-12103 Berlin, Germany
2Department of Clinical Biochemistry, Copenhagen University Hospital (Roskilde) and Roskilde Hospital, 7-13 Køgevej, DK-4000 Roskilde, Denmark
3Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark
†These authors contributed equally to this work
Accepted 11 October, 2013; Available Online 29 October, 2013
Background: Diagnosis of lactose intolerance is commonly based
solely on the absence of the European lactose tolerance allele
MCM6 -13910T, although other lactose tolerance mediating alleles
exist, especially in defined African populations and in the Middle
East. Therefore, a SimpleProbe assay was developed which can
simultaneously detect -13910T as well as common African variants.
Methods: A SimpleProbe oligonucleotide was used to discriminate between three classes of MCM6 alleles: I: Intolerance (-13910C); II: Caucasian type tolerance (-13910T); III: African/Arabian type tolerance (-13907G, -13913C, -13915G). Genotyping standards for all these alleles were constructed through PCR amplification and cloning from genomic DNA. Assay performance was tested using real-time PCR and melting curve analyses with genotyping standards as templates. Finally, the genotyping accuracy was tested using two panels: 100 individuals, previously genotyped using Sanger-sequencing and 20 individuals, previously genotyped using TaqMan analyses.
Results: The assay showed a clear discrimination between gene variants conferring lactose tolerance and intolerance. Mean Tmvalues for the Caucasian alleles -13910C and -13910T were 55.3 °C and 60.8 ºC respectively, whereas Tm-values for all three African/Arabian variants (-13907G, -13913C and -13915G) were in the interval of 50-52 ºC. SimpleProbe genotyping of both panels showed 100 % concordance with the results of the reference methods.
Conclusions: The new SimpleProbe LightCycler assay provides a rapid, cost effective and reliable method to diagnose lactose intolerance and is especially suited for populations with African or Middle Eastern admixture.
Keyword:lactose tolerance, MCM6 gene, African variants, SimpleProbe LightCycler assay