Presented at the Neonatal Society 2004 Autumn Meeting .
Gavriel G, Stanier P, Moore GE, Modi N
Imperial College London, UK
Background: Taking blood from newborn infants as a means of obtaining DNA for research purposes is an invasive procedure and is considered both unethical and undesirable. Sufficient human DNA for direct gene analysis can be isolated from adult buccal epithelial cells (1). A similar approach in infants would be of value but to our surprise we found no relevant published data.
Aim: We aimed to determine the ease and feasibility of isolating neonatal buccal cells for DNA extraction followed by PCR and sequence analysis.
Methods: The study was approved by the Riverside Research Ethics Committee and written, informed parental consent was obtained. Buccal cells were collected from term and preterm babies on the neonatal unit or postnatal wards at Chelsea & Westminster Hospital. A microbiological swab or cotton dental roll was rubbed on the inner cheek and the baby was allowed to suck on it for 30 seconds. Swabs/dental rolls were placed in 0.9% sodium chloride solution to release the cells and then centrifuged to concentrate the cells into a pellet. The swab/dental roll and the sodium chloride solution were removed and the cells lysed with a standard lysis buffer. DNA was then extracted using a simple phenol-chloroform technique (2). Spectrophotometry was carried out and DNA concentration and yield calculated from UV absorbance at 260 nm (A260). To test the integrity of the DNA, PCR was carried out to amplify exon 22 of the human SCRIBBLE gene (SCRB1), which has been associated with severe neural tube defects in the mouse (3). PCR products were viewed on a 1% agarose gel with a UV transilluminator. PCR products were sequenced using an automated Sanger dideoxy method. Sequence alignment with the BLAST program was used to confirm accurate sequence amplification.
Results: Buccal cell samples were obtained from 8 term and 4 preterm infants. The infants tolerated the procedure well and appeared unperturbed by it. Mothers found it very acceptable. Spectrophotometry confirmed successful DNA extraction from all samples with a mean DNA yield of 6.65 g (range 1.65-16.40). There was no difference in yield between swabs and dental rolls but the former were easier to handle in the laboratory. A region of SCRB1 was successfully amplified by PCR from all 12 buccal cell samples. Sequences matching exon 22 of SCRB1 were successfully obtained using both forward and reverse primers.
Discussion: This work has demonstrated the feasibility of obtaining neonatal buccal cells in sufficient quantity for DNA extraction, PCR and sequencing analysis. The DNA yield obtained compares favourably with similar studies with adult and child subjects where yields ranging from 2 – 85 μg have been described. The technique is simple and non-invasive and removes the need for blood sampling. These findings are of potential importance in relation to neonatal research. It will also be of interest to establish the reliability and reproducibility of the technique in the context of the diagnostic laboratory.
References
1. Lench N, Stanier P, Williamson R. Lancet 1988; 1(8599):1356-1358. 2. Kunkel LM, Smith KD et al. Proc Natl Acad Sci U S A 1977; 74(3):1245-1249. 3. Murdoch JN, Henderson DJ et al. Hum Mol Genet 2003; 12(2):87-98.