Dried neonatal blood, stored and processed on filter paper, has been the standard for neonatal screening for 50 years [1
]. The ordinary use of neonatal blood is based upon the excision of blood spot punches, typically 3 mm-6 mm in diameter, followed by physical or biochemical analysis of serum analytes released from the punch by soaking in alcohol or water [2
]. More recently, dried blood spots have been used to screen for heritable traits at the DNA level, typically traits such as cystic fibrosis and the thalassemias, and other traits that are readily assayed by PCR tests [3
In 2005, based on recent advances made in highly parallel microarray technology, geneticists at March of Dimes proposed that we may be entering an era where the DNA complement of such dried blood spots might be sufficient, in terms of quantity and quality, to support genome-wide analysis of complex heritable traits, thereby leapfrogging the limits of single-gene analysis[5
In spite of the exciting prospects implied by that 2005 review, relatively little work has been published in the intervening three years to validate such genome-scale neonatal screening [6
]. Microarray technology has improved significantly in that period, in terms of diminished cost and sample requirement, and has yielded increased data density and quality [8
]. However, such genome-scale microarray analysis continues to require an input DNA mass (about 250 ng) that is about 100 times larger than required for simple PCR testing; requires DNA that is double stranded; and requires DNA with a length-span that is about 5 times longer than required for most PCR reactions. Thus, going forward, it is suggested here that a major technical barrier to the adoption of genome-wide microarray technology may not be the microarray technology per se, but instead, may be the quantity and quality of DNA that can be usefully recovered from an ordinary air-dried neonatal blood specimen.
The importance of DNA recovery from such Guthrie cards is discussed at length in a recent comparative study by Sjoholm and colleagues [9
]. They have compared a number of commercially available kits and procedures for DNA recovery from Guthrie cards and have show that only about 15%–25% of the total DNA complement can be recovered. They have measured DNA recovery from dried blood spots stored for up to 26 years, and have shown that, on standard 3 mm punches from such cards, DNA yields (with the best available technology) are only about 30 ng per punch.
However, in spite of the relatively low yields, Sjoholm have shown that the small amount of DNA obtained remains an excellent substrate for whole genome amplification, and relatively complex multiplex SNP analysis [9
]. However, for genome wide scanning methods such as microarray analysis (which require at least 250 ng of input DNA) the relatively low DNA recoveries, obtained by Sjoholm, would require extraction and pooling of as many as eight 3 mm punches: a value that is difficult to reconcile for such rare specimens.
Generally similar results have been obtained by Mas (10) in a study of dried blood spots stored on treated filter paper matrices such as Whatman FTA or IsoCode, employing the manufacturer's extraction method. In that study, about 25% recovery was obtained in a single extraction, to yield up to 150 ng of single stranded DNA as a 200 uL solution, per 40 uL of adult human blood input [10
]. As for the by Sjoholm, the DNA obtained by Mas et. al. could be used effectively for multiplex PCR and for whole genome amplification, but as the authors correctly noted, might be too dilute too support more complex studies such as genome wide microarray analysis. Moreover, since the DNA extraction procedures employed by Mas yielded denatured DNA, the product of such extractions would not be applicable to methods such as Affymetrix microarrays, which require that the DNA substrate remains in a native, double stranded state prior to analysis.
Here, we describe the use of a new technology, referred to as GenSolve™, originally developed as a high-efficiency method to recover native DNA from blood spots on chemically treated FTA filter paper [11
] but used here to recover DNA from neonatal blood spots on standard Guthrie cards collected from 1991 to 2003 as part of the California Birth Defects Monitoring Program. The GenSolve technology was used in combination with standard DNA purification, followed by analysis on the Illumina 610 bead array microarray platform, which interrogates about 610,000 sites of human SNP variation in parallel [12
Although the Illumina 610 chip does not contain content that was developed specifically for neonatal screening, the scale of the analysis performed on the Illumina 610 chip can be viewed as a technical surrogate for any large panel of genome-wide SNP testing that could be developed as a screening tool. For the immediate future, neonatal screening will likely continue to employ biochemical analysis mediated by tandem mass spectrometry and related methods, to which genetic testing will be added, in parallel. Typically, only a small fraction of a dried neonatal sample will be available for microarray or microarray-like genetic analysis. Thus, the work described here is focused on microarray testing using DNA obtained from only two 3 mm diameter Guthrie card punches, roughly 1/40th
of the blood ordinarily collected from a neonate, on a standard 5-spot Whatman 903 Guthrie card [13