Tools For DNA Diagnostics (July 1995)

Arrayed Primer Extension (APEX): The Next Generation DNA Analysis System for Sequencing in DNA Diagnosis

Sponsor: Amersham Biosciences Corp. (formerly USB/Amersham Pharmacia Biotech, Inc.)

• Project Performance Period: 9/15/1995 - 9/14/2000
• Total project (est.): $11,597,709.00
• Requested ATP funds: $5,785,406.00

Pharmacia Biotech, Inc., and its partners in this joint venture propose to develop the concept of "arrayed primer extension" into a routine, cost-effective, DNA sequencing system primarily for screening, diagnosing, and monitoring known genetic disorders. The technique is generally applicable to the task of resequencing genetic information, particularly where this must be done repeatedly. The strategy is to create a large array of short, single-stranded oligonucleotides (attached to a substrate) of known sequences to which complementary strands of DNA in the sample will attach. At those sites where attachment occurs, a DNA polymerase enzyme will add a fluorescently labeled nucleotide to the oligonucleotide. Then, by detecting and analyzing the resulting fluorescence pattern from the array of oligonucleotides, the sequence of the DNA sample can be inferred. Advantages claimed for this technique over usual hybridization strategies are reduced mismatching due to the intercession of the polymerase, increased resistance to oligonucleotide failure sequences, tolerance of a greater range of hybridization conditions, and lower background. Major tasks of the project are developing efficient chemical strategies for synthesizing oligonucleotides (both in situ and ex situ), bonding the oligos into arrays on glass slides or other substrates, developing a quartet of fluorescently labeled nucleotides (each corresponding to one of the four DNA building blocks) that a polymerase enzyme will be able to incorporate into the oligonucleotides, developing a fluorescent imager capable of detecting the wavelengths of all four dye molecules, and writing software that can infer the sequence of the DNA in diagnostic samples from the fluorescence pattern of the array.The joint venture's partners include Baylor College of Medicine (Houston, TX), Duke University (Durham, NC), Identigene, Inc. (Houston, TX), and BioImage Systems Corporation (Ann Arbor, MI).

For project information:

Dr. Leslie C. Beadling, (908) 457-8071

Active Project Participants

• Baylor College Of Medicine (Houston, TX)
  [Original, Active Member]
  
• Duke University, Department of Chemistry (Durham, NC)
  [Original, Active Member]
  
• Identigene, Inc. (Houston, TX)
  [Original, Active Member]
  

ATP Project Manager

Douglas Bischoff, (301) 975-8597
douglas.bischoff@nist.gov