Use of Pyrosequencing for the Detection of Several Polymorphisms Associated with Lactase Persistence
Catherine Herrera1, Gunnhild Kravdahl1,*, Malene Nilsen1
1Multidisciplinary Laboratory Medicine and Medical Biochemistry, Akershus University Hospital, Oslo, Norway
Introduction: The single nucleotide polymorphism (SNP) -13910C>T is strongly associated with lactase persistence (LP) in Caucasian from northern Europe. In other ethnical groups, LP is associated with other SNPs such as -13907C>G, (Ethiopian/Sudanese), -13915T>G (Arab) and -14010G>C (Kenyan/Tanzanian). In Norway, -13910C>T-genotyping is widely and increasingly used in the diagnosis of primary lactose intolerance (LI), but this analysis is of limited value in patients of non-European descent. To improve the diagnostic performance in multiethnic populations, several laboratories have established methods based on real-time PCR to detect SNPs other than -13910C>T. Though these methods are reliable, some obstacles might be encountered when designing primers/probes. Hence sequencing is a better alternative. Sanger sequencing is time-consuming and laborious and today’s laboratories are therefore in need of a sequencing method with less hands-on time.
Objectives: The aim was to create a lean workflow by using pyrosequencing for genotyping of both well-documented and other possibly LP-associated SNPs that can be encountered within the MCM6-region.
Methods: 83 DNA-samples with SNPs in various positions of the MCM6-region were sequenced using the Pyromark Q24 (Qiagen) and AB3130 (Life Technologies). The primer and probe sequences were based on the work of Nilsson et al (2008). New dispensation orders had to be created to detect all SNPs.
Results: The new dispensation orders made it possible to detect all the SNPs in the vicinity of position -13910 in the MCM6-region making the interpretation easier. There were no discrepancies between the results from the Q24 and AB3130.
Conclusion: By combining the Taqman method for genotyping of -13910C>T and pyrosequencing, it is possible to customize the genotyping for lactose intolerance according to ethnicity, without affecting the response time and using expensive resources. The hands-on is less extensive compared to Sanger resulting in a user-friendly workflow.
Keywords: DNA, Method