Development of New Direct DNA Sequencing Method

  Winston Chen and his colleagues in the Life Sciences Division at ORNL recently developed a new direct DNA sequencing method by ultraviolet matrix-assisted laser desorption/ionization/fragmentation time-of-flight mass spectrometry (MALDIF). With this approach, direct sequencing by physical methods without the need of DNA ladders preparation has been successfully demonstrated. The sequencing time for each short ss-DNA sample was reduced to be less than one second compared to several hours for conventional gel electrophoresis.

  For conventional gel electrophoresis DNA sequencing method, three major steps are needed. They include:

1. DNA ladders preparation by either Sanger's enzymatic method or Maxam-Gilbert chemical degradation method. This step takes about 10 minutes.
    
2. Running gel to separate different sizes of DNA. This step typically take several hours.
    
3. Detection of each size of DNA. For fluorescence detection method, this step is finished right after DNA separation. However, for radioactive tagging, this step can take another several hours.

  During the past 5 years, several groups have tried to use laser and mass spectrometry for DNA sequencing. There are three possible different approaches to sequence DNA.

1. Use of mass spectrometer just as a better detector. With this approach, both preparation of DNA ladders and running gel for DNA separation are needed.
    
2. Use of matrix-assisted laser desorption/ ionization for DNA sequencing. With this approach, the laser energy is absorbed by small matrix molecules which vaporize and carry DNA molecules into space for ionization and detection. Thus the fragmentation of DNA can be reduced to minimum. With this approach, gel electrophoresis can be eliminated. The time for separation of different sizes of DNA is reduced from several hours to less than one second. However, DNA ladders preparation is still necessary. Thus the overall time saving is from several hours to about 10 minutes. Almost all mass spectrometry sequencing groups at the present time take this approach.
    
3. Use of laser ablation to achieve desorption, ionization, and selective fragmentation. With this approach, DNA ladders are produced during the laser ablation process. Thus, DNA ladders preparation, separation of different sizes of DNA and detection of different sizes of DNA are all accomplished in less than one second. Thus, the overall time saving is from several hours per sample down to less than one second.

  In addition to time-saving, approaches (2) and (3) also need no tagging of either radioactive materials or hazardous chemical dyes.

  By discovering new matrices for selective cleavage of DNA between bases, ORNL researchers have succeeded in using approach (3) in sequencing blind short ss-DNA with the size up to 35 nucleotides. Thus, this method can be directly used for sequencing primers, which is extremely difficult to achieve by any other methods. If this approach can be extended to longer oligonucleotide, it should have a revolutionizing impact on Human Genome Project, Disease Diagnosis, and DNA Finger Printing for Forensic applications.

Contact: Winston Chen
Telephone: 423-574-5895
E-mail: che@ornl.gov