Participants
The Vermont Family Study is a collection of samples from 207 families comprising 783 individuals. 167 families were part of an ADHD sample with one member of each family recognized as a Proband for the disorder. 40 families were included as control families with no DSM-IV diagnoses.
Sample Collection
Participants were asked to refrain from eating or drinking 1 hour prior to saliva collection. Each individual was instructed to place a standard 2" × 2" piece of cotton gauze in the buccal region of their cheek for 3 minutes. The saliva saturated cotton spit wad was removed, rolled to fit a collection tube, and stored at -20°C until the genomic DNA was extracted. All data collected and analyzed with approval of the UVM COM IRB Ethics Committee.
DNA Extraction and Quantification
All DNA extractions were performed at the Avera Institute for Human Behavioral Genetics. DNA was extracted from saliva saturated cotton spit wads using a column-based purification method. Rolled spit wads measured approximately, 5 cm × 1.5 cm, and DNA was extracted from buccal cells using the QIAamp DNA Blood Midi Kit large volume protocol (Qiagen) according to the manufacturer's instructions with a few modifications. The spit wad was incubated at 70°C in a Protease/Lysis buffer mixture (200 ul Qiagen Protease/2.4 ml buffer AL) for 30 minutes in a 15 ml conical tube (Fisher Scientific). The lysate was separated from the spit wad using centrifugal force by placing the spit wad in the barrel of a 5 ml syringe (Becton Dickson) that was seated in a conical 15 ml tube and centrifuged for 10 minutes at 12,000 rpm. The spit wad was discarded and 2 ml absolute ethanol was added to the lysate and the tube was mixed by vigorous shaking (vortexing). Approximately one half of the lysate/ethanol mixture was transferred to a Qiagen Midi column placed in a clean 15 ml conical tube. The column was centrifuged @ 1850 × g for 3 minutes. The filtrate was discarded and the remaining lysate/ethanol mixture was applied to the same column and centrifuged @ 1850 × g for 3 minutes. The column was washed with 2 mls of buffer AW1 and centrifuged @ 3220 × g for 2 minutes. The column was washed a second time with 2 mls of buffer AW2 and centrifuged @ 3220 × g for 30 minutes to ensure complete drying. DNA was eluted from the column into a clean 15 ml conical tube by adding 200 μl buffer AE to the column, incubating at room temperature for 5 minutes, and centrifuging @ 3220 × g for 4 minutes. For maximum DNA yield a second elution was performed as described above, yielding approximately 400 μl total volume. DNA concentration and purity were determined using UV spectrophotometry (Nanodrop). All genomic DNA was either diluted or concentrated to a final concentration of 50 ng/ul. DNA was diluted in a reduced EDTA buffer (10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0) and concentrated using Microcon YM-100 centrifugal filter devices (Millipore) according to the manufacturer's instructions.
PCR Amplification
The Quality of DNA isolated from the spit wad was assessed by PCR amplification of the serotonin-transporter-linked polymorphic region (5HTTLPR). PCR products were visualized using 2% agarose gel electrophoresis or fragment analysis on an ABI 3130 Genetic Analyzer (Applied Biosystems, Inc.). Primer sequences for 5-HTTLPR were previously described; forward primer (5' -ATGCCAGCACCTAACCCCTAATGT-3') and the reverse primer (5'- GGACCGCAAGGTGGGCGGGA-3') [7]. When running samples on the 3130 genetic analyzer a fluorescently tagged forward primer (6FAM, Applied Biosystems) was used to tag the PCR product for fragment analysis. This primer pair amplifies a 419 base pair product for the 16-repeat long (L) allele and a 375 base pair product for the 14-repeat short (S) allele. PCR reactions were performed using a PCR Master Mix (Promega) containing a final concentration of 1.5 mM MgCl2, 1× reaction buffer, 200 μM of each dNTP, 40 ng purified genomic DNA, 1.25 units Taq DNA polymerase, and 5 pmols of each primer in a 25 ul reaction. PCR cycling conditions consisted of an initial denaturation at 95°C for 15 minutes, 35 cycles each consisting of 30 s at 94°C, 30 s at 66°C, and 40 s at 72°C. Elongation was continued for 15 min at 72°C after the last cycle. S vs. L fragments were called using GeneMapper Software Version 4.0 (ABI), or fragments were separated on a 2% agarose gel supplemented with ethidium bromide (0.02%, Fisher).