Margaret C. Kline, Janette Redman and Dennis J. Reeder
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD
For the past three years, researchers at NIST have served in a quality assurance capacity for the Armed Services DNA Repository/DNA Identification Laboratory. Each month, 100 samples consisting of duplicate one-eighth inch punches from bloodstained cards are taken from a randomized repository specimens. These bloodstained cards have been stored no longer than five years at -20°C. All samples are extracted at NIST using a standard Chelex method and the DNA-containing supernates are typed by any one of several available genetic tests. Such tests include D1S80, AmpliTypeŽ HLA DQalpha, AmpliTypeŽ PM, HUMTH01 or HUMVWFA31. We are expanding our battery of test methods to include: HUMCSF1PO, HUMF13A01, HUMFESFPS, and HUMTPOX. Along with the above monoplex systems, protocols for multiplex systems are also being developed to include the CTT (GenePrint™ STR System that includes CSF1PO, TPOX, and TH01), and a quadruplex system (HUMFESFPS, HUMFA01, HUMTH01 and HUMVWFA31) for the Perkin-Elmer ABD GeneScanner.
Our studies have shown that, as expected, there is a 100 percent correlation between the typing results achieved with the monoplex manual STR systems and the automated STR systems. However, we lose the ability to differentiate the HUMTH01 9.3, 10 genetic type with the automated system.
Starting quantities of DNA ranging from 0.5 to 14 ng were successfully amplified in both the manual monoplex and the quadruplexed automated systems. Amplification differences were found to be independent of the quantity of input DNA. Further, a preferential amplification of the HUMF13A01 alleles was observed in three cell lines included as controls. These studies indicate that methods development should be conducted with a wide variety of individuals and should not rely solely on cell line DNA.
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