[Standardization of DNA extraction with NaCl from oral mucosa cells: application in PROP1 gene study].
DNA extraction of peripheral leukocytes is the most broadly used technique to obtain DNA. However, cell collection from an oral swab, frequently used in forensics, is useful to obtain DNA samples, mainly in newborns, children and patients who live far from the collection sites, where blood sample collection and sending is not feasible. Our objective was to standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit. To test DNA quality, we amplified the 3 exons of PROP1 gene of 12 patients with hypopituitarism in DNA obtained from oral cells and peripheral blood cells. Amplification of larger fragments was tested in oral DNA of normal subjects using primers of exon 10 of FSHR gene (1000 bp) and of CYP21A2 gene (1200 bp). Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared to the commercial kit. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In conclusion, we standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that DNA from oral swabs are a reliable source for genetic studies.
Laboratório de Hormônios e Genética Molecular-LIM/42, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, SP.
Milena Garcia
MG
Abrão
Ana Elisa C
AE
Billerbeck
Mirian Yumie
MY
Nishi
Suemi
S
Marui
Berenice B de
BB
Mendonça
D016022:Case-Control Studies; D002648:Child; D004247:DNA / Q000302:isolation & purification*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D005784:Gene Amplification; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007018:Hypopituitarism / Q000235:genetics*; D007231:Infant, Newborn; D007962:Leukocytes / Q000737:chemistry; D009061:Mouth Mucosa / Q000166:cytology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012965:Sodium Chloride
D018398:Homeodomain Proteins; C102134:Prophet of Pit-1 protein; D012965:Sodium Chloride; D004247:DNA