DNA Molecules as Standard Reference Materials I: Development of DNA Identification Sequences and Human Mitochondrial DNA Reference Sequences

This paper describes the design, construction and characterization of two DNA molecules that function as prototype Standard Reference Materials for use in the determination of DNA sequences. The first prototype reference DNA molecule was constructed to function as a true reference DNA sequence encoding the human mitochondrial hypervariable region 1. The second DNA molecule was designed to be a molecular DNA “tag” for use in the identification of DNA Standard Reference Materials. DNA molecules were chemically and enzymatically synthesized, cloned in molecular cloning vectors and sequenced using manual and automated fluorescent DNA sequencing methods. The sequence of the cloned human mitochondrial HV1 DNA shows six base pair differences from the original published DNA sequence. The molecular DNA tag sequence uses the universal genetic code with one letter amino acid abbreviations to spell the words “NIST DNA STANDARD REFERENCE MATERIAL.” The need and utility of an expanded molecular code to uniquely identify DNA molecules is discussed. The use of these DNA molecules in the development of DNA Standard Reference Materials is also described.