Utility of PCR for DNA analysis from dried blood spots on filter paper blotters.

T h e collection of dried blood spots on filter paper blotters is a major innovation and has facilitated the development and dissemination of newborn screening programs principally because of the ease of sample collection, handling, and shipment to centrally located laboratories and the stability of the analytes in these specimens. (1,2) These samples, which are also known as spots, after their originator Dr. Robert Guthrie, have been used for a considerable number and a broad variety of screening tests. Examples of these tests are shown in Table 1, organized according to newborn screening detection categories. The traditional analyte categories that have been targeted by newborn screening programs have been metabolites, hormones, and proteins (Table 1). (2,3) This report will show that DNA represents a new analyte category for direct mutation detection from these Guthrie spots. In addition to describing how analysis of polymerase chain reaction (PCR)-amplified DNA is beginning to interface with neonatal screening programs, we will discuss the use of dried blood specimens for determination of mutant allele frequencies in various populations, as well as for forensic identification and databases, infectious disease diagnosis, and large animal agriculture. The ability of this PCR-based molecular genetic technology to provide large-scale screening and diagnosis will require the development of automation to reduce the number of personnel needed, and we also will consider current and future developments in this area. In 1987 it was reported that DNA could be obtained from the dried blood specimens on filter paper blotters. (,4) The initial efforts resulted in microextraction of approximately 500 ng of DNA from a single 1/2-in-diameter specimen circle representing the dried equivalent of approximately 50 ~1 of whole blood. This amount of DNA was sufficient for a single Southern blot analysis, but DNA in these amounts was approaching the limits of the existing technology. Fortunately, as these original investigations were being completed, the technique of PCR amplification was reported/s) and it was shown that the DNA obtained from the dried blood spots could be amplified by PCR. (6) Improvements in the microextraction technique permitted isolation of DNA from as little as a 1/8-indiameter punched disc, representing the dried equivalent of approximately 3 lal of whole blood. (6) The development of the heat-stable polymerase from Thermus aquaticus (7) and a programmable heating block (8) permitted automated temperature cycling to replace the previously tedious manual PCR procedures.