Stability of genomic DNA in dried blood spots stored on filter paper.

The stability of DNA in dried blood samples obtained from the neonatal screening program in Thailand was retrospectively studied in order to determine the conditions necessary for the long term storage of samples for DNA banking. Specimens from 1991 to 2001, which had been kept in the ambient conditions at the Department of Medical Science, Ministry of Public Health, Thailand, were randomly sampled and used for the study. Genomic DNA was extracted from the samples and DNA fragments of the PAX8 and beta-globin genes were amplified by PCR to determine DNA stability. The study showed that 255-bp and 674-bp fragments of the PAX8 gene could be amplified from all the samples. The DNA fragment of 1,039 bp of the beta-globin gene could be detected in all of the samples for the years 1993 to 2001, but only in seven and five out of the ten studied samples for each of the years 1991 and 1992, respectively. Our study shows that genomic DNA is stable in dried blood stored on filter paper at ambient tropical conditions for at least 11 years. However, DNA quality for amplification of larger DNA fragments decreased when the specimens were stored for longer than 10 years.

A simple method for extraction and purification of genomic DNA from dried blood spots on filter paper.

We have developed an efficient and simple method for extracting and purifying genomic DNA from dried blood stored on filter paper. The quality of the genomic DNA extracted is tested by PCR amplification of a 255-bp fragment of the PAX8 gene sequence and the PCR products are determined for further genetic studies by single strand conformation polymorphism (SSCP) analysis. Larger DNA sequences of the 674-bp of the PAX8 gene and the 1,039-bp of the human beta-globin gene, a housekeeping gene, have also been amplified from the extracted DNA, thus indicating the high quality of the genomic DNA extracted by the developed method for subsequent genetic studies of any gene of interest. The method developed can also be used for the purification of genomic DNA from dried blood specimens stored under different conditions. Moreover, the genomic DNA products can be stored for long-term use due to the highly purified procedure. Therefore, the method is efficient and appropriate for the extraction and purification of genomic DNA from dried blood specimens, which has become an increasingly important tool for genetic and epidemiological studies.

A study to implement early diagnosis of HIV infection in infants born to infected mothers.

A protocol for detecting HIV DNA from specimens collected on filter papers and the effect of storage temperatures on determination of HIV DNA from dried blood spots has been developed and optimized. Blood specimens collected from HIV-1 infected and normal persons were spotted onto blood collection cards (Whatman BFC 180). The HIV DNA was extracted by phenol-chloroform-isoamyl alcohol and was detected for C2V4 of HIV-1 env by nested polymerase chain reaction (nested PCR). One set was stored at -20 degrees C for 14 weeks, another at 37 degrees C for 1 week and then kept at -20 degrees C for 13 weeks and a third set at 25 degrees C for I week and then -20 degrees C for 13 weeks. The dried blood spots from each set were detected for the HIV DNA every 2 weeks for 14 weeks. The C2V4 region of HIV env DNA was determined from small amounts of the dried blood collected on the filter papers. The nested PCR procedure could detect as few as 5 copies of HIV proviral DNA, and HIV DNA could be detected from specimens with viral loads of 2x 10(4) copies/ml. HIV DNA could be detected from specimens collected at all temperatures tested for at least 14 weeks. Therefore, laboratory diagnosis of HIV infection can be done by PCR on dried blood spots. These techniques will be useful as a tool for studying the epidemiology of HIV infection among populations of interest such as mother to child infection using newborn screening specimens.