The DNA Measurements Group focuses on research efforts to meet goals and objectives in areas of measurement science, standards and data dissemination. Projects include the measurement of DNA damage and DNA repair, the characterization of DNA including mutation detection and genetic toxicology, human identity profiling, and the development of DNA Standard Reference Materials (SRMs).

In the area of DNA diagnostics, the group has established the NIST-Early Detection Research Network (EDRN) Biomarker Validation Project, an integral part of a large-scale effort of the National Cancer Institute to develop new biomarkers for early cancer detection. In related work, background research is underway to provide a new standard reference material (SRM) for HER2 testing in breast cancer patients. This will focus on development of a substrate for assays based on fluorescent in-situ hybridization (FISH) and immunohistochemical (IHC) technologies. Such assays that preserve cellular architecture are being adapted to automated, high throughput platforms with chemistries that incorporate quantitative fluorophores such as semiconductor nanocrystals (quantum dots). In addition, we are developing improved high-throughput measurement methods and a standard reference material for telomerase. This will provide the means for clinical evaluation of telomerase as a cancer biomarker.

The group’s efforts also include the study of the mechanisms of oxidative stress, DNA damage and actions of DNA repair enzymes. Oxidative stress is produced in cells by oxygen-derived species including free radicals that result from cellular metabolism and from interaction with cells of carcinogenic compounds, redox-cycling drugs and ionizing radiations. Free radicals cause DNA damage and are associated with numerous diseases such as cancer, heart disease, and Alzheimer’s disease. This work includes the development of methodologies for the measurement of DNA damage and repair in cells. Using techniques that include gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry, the group has an ongoing program on the characterization of DNA damage and the functions of numerous DNA repair enzymes in living cells. DNA repair enzymes are essential for preserving the integrity of the genetic material and are potential anti-viral and anti-cancer drug targets. Accuracy and traceability among laboratories is addressed by the development of an SRM consisting of stable isotope-labeled modified DNA bases to be used for mass spectrometric measurements .

In the area of human identity/forensic science, the group focuses on performing research for rapid determination of DNA profiles by polymerase chain reaction (PCR) amplification and automated detection of fragments. Efforts are also made in the development and evaluation of high throughput technologies for typing single nucleotide polymorphisms (SNPs) for human identification purposes. In addition, the group studies cooperative development of short tandem repeat (STR) and SNP detection technology, multiplex PCR, DNA stability studies, Y chromosome markers, mitochondrial DNA sequencing, and attendant standards. Techniques include sensitive staining of electrophoretic gels, use of chemiluminescence, enhanced applications of capillary electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In an effort to support the growing demand for Y chromosome testing, efforts include standards that can be used to calibrate instrumentation and verify assay performance with Y STRs and Y SNP markers. In the area of data dissemination, the NIST Web-based forensic database for STRs can be accessed at http://www.cstl.nist.gov/biotech/strbase.

Other efforts include providing the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders such as Fragile X syndrome. Work focuses on the development of an SRM to be used to detect the correct number of triplet repeats in Fragile X patients and their relatives. The group also studies the design and use of different techniques for detecting heteroplasmic low-frequency mitochondrial SNPs and mutations. Techniques include denaturing gradient gel electrophoresis, the Luminex 100 system and the use of peptide nucleic acids (PNAs). The PNA technique can detect the single nucleotide mitochondrial heteroplasmic mutation associated with the disease Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like Episodes (MELAS) in asymptomatic or symptomatic carriers with low to undetectable blood levels (0.1%) of this mutation. The development of a heteroplasmic mitochondrial SRM is underway to allow investigators to determine the sensitivity of their mutation detection techniques. Information on mitochondrial mutations can be found at www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html.