Development and validation of the high-quality 'rapid method for swab' to genotype the HTTLPR serotonin transporter (SLC6A4) promoter polymorphism.
BACKGROUND
The importance of genetic variation to the etiology of neuropsychiatric disorders is well established and is currently being examined for diagnosis and treatment. The most popular method of obtaining material for genotype analysis, high-yielding DNA extraction from blood, has several limitations, including invasiveness, need for skilled individuals to collect material, and requirement for cold storage. Saliva sampling is noninvasive and trained personnel are less necessary, but it still requires a relatively high level of subject compliance. Buccal mucosa cells sampling is almost completely noninvasive, reducing compliance issues significantly. Samples collected have been shown to produce usable DNA after shipment through conventional mail. The DNA produced by rapid elution of these swabs in chaotropic buffers is, however, of limited quality and low purity.


OBJECTIVE
Our aim was to develop a rapid, economical, and environmentally safe method for extraction of high-quality genomic DNA, which can be used to determine clinically important genotypes from trace quantity samples and which has sufficient yield for multiple assays.


METHODS
We developed a method of extracting high-quality genomic DNA from buccal swab, which we termed the 'rapid method for swab' (RMS). We compared RMS with two established procedures, specifically the original rapid method and the commercially available Buccal Amp method. We assessed the generated genomic DNAs by their (i) quality, (ii) quantity, (iii) restriction enzyme digestibility, and (iv) PCR-based genotyping in addition to time, cost, and environmental impact of the procedures.


RESULTS
DNA generated by RMS was of higher purity than that by Buccal Amp. RMS is nonenzymatic and does not use strong chaotropic salts or extreme pH. We also showed the suitability of RMS-DNA for LA/LG genotyping as generated by PCR using 7-deaza-dGTP.


CONCLUSIONS
The RMS procedure is novel, efficient, safe, and yields sufficient material for multiple genotyping analyses. The RMS produces DNA of high quality from a single human buccal swab. RMS is a noninvasive technique and particularly suitable for children and older individuals and in field collection settings.
Maloney
Bryan
B
Laboratory of Molecular Neurogenetics, Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN-46202, USA.
Ray
Balmiki
B
Hayden
Elizabeth P
EP
Nurnberger
John I
JI
Lahiri
Debomoy K
DK
D004247:DNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D005838:Genotype; D006801:Humans; D008970:Molecular Weight; D009061:Mouth Mucosa / Q000166:cytology; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics*; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D013057:Spectrum Analysis; D013997:Time Factors
C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D004247:DNA