1 3
Cancer Chemother Pharmacol
DOI 10.1007/s00280-015-2729-4
SHORT COMMUNICATION
Validation of a fast and low-cost alkaline lysis method for gDNA
extraction in a pharmacogenetic context
Ruth Ramos-Díaz
1
· Fernando Gutiérrez-Nicolás
1
· Gloria J Nazco-Casariego
1
·
Itamar González-Perera
1
· José A Pérez-Pérez
2
Received: 23 January 2015 / Accepted: 16 March 2015
© Springer-Verlag Berlin Heidelberg 2015
Introduction
The importance of human genetic variability on drug effi-
cacy and safety has been highlighted in the last years [1].
This progress on pharmacogenetic knowledge, together
with genotyping assays becoming cheaper, has enabled the
potential incorporation of all this innovation into daily clin-
ical practice [2]. One of the short-term challenges faced by
personalized medicine is ensuring that genetic analysis, and
more specifically pharmacogenetics, forms part of services
portfolio in most hospitals, providing valuable information
to the clinical practitioner for selecting the most appropri-
ated drug therapy for patients [3].
The first step towards determining a patient’s genotype
is to obtain genomic DNA (gDNA) from a biological
sample, whose source will depend on the kind of genetic
variation under study: for the analysis of somatic muta-
tions associated with cancer, the starting sample must
contain tumour cells, which normally requires perform-
ing a tumour biopsy [4]. However, when the goal is to
identify germline mutations, a simple peripheral blood
test is sufficient. In this latter case, the use of dried blood
samples (DBS) is increasingly common, not only because
of its simple handling and transport, but also because of
the possibility of storage for several years. Several pro-
cedures for extraction of gDNA, from DBS, have been
published. Among them, a method based on alkaline
lysis reported by Rudbeck and Dissing [5] is especially
attractive because of its simplicity, speed and low cost.
Nevertheless, its use has not been generalized in clinical
laboratories. In this work, we have assessed the suitabil-
ity of the gDNA obtained with this method in the con-
text of pharmacogenetic analysis by evaluating its ability
to meet the quality requirements of different genotyping
techniques.
Abstract
Purpose Translation of pharmacogenetic findings from
the research laboratory to the clinical practice demands
simple and efficient procedures. In this sense, we evaluated
the suitability of a modified protocol for genomic DNA
extraction based on alkaline lysis of cells.
Methods Dried blood samples were obtained from 48
patients diagnosed with colorectal cancer. A total of 11
mutations in the dihydropyrimidine dehydrogenase gene
and related to 5-fluorouracil toxicity were searched by
amplicon sequencing and real-time PCR with fluorescent
probes.
Results Genomic DNA extracted with the alkaline lysis
method, both from dried blood samples and buccal swabs,
fulfilled the quality requirements of the two genotyp-
ing methods assayed, which yielded 100 % concordant
results for 11 genetic variants with relevance to cancer
chemotherapy.
Conclusions The assessed protocol has shown to be a
very fast and economical approach to perform genetic
analyses in the clinical laboratory for pharmacological
purposes.
Keywords DNA extraction · Dried blood samples ·
Buccal swabs · Pharmacogenetics
* Fernando Gutiérrez-Nicolás
fgunico@gmail.com
1
Pharmacy Department, University Hospital of the Canary
Islands (HUC), Tenerife, Spain
2
Biochemistry, Microbiology, Cell Biology and Genetics
Department, University Institute of Tropical Diseases
and Public Health of the Canary Islands (IUETSPC),
University of La Laguna, Tenerife, Spain