45. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis 

C. H. Winston Chen, N. R. Isola, N. I. Taranenko, V. V. Golovlev, and S. L. Allman 
Life Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 
chenc@ornl.gov 

During the past few years, rapid progress has been achieved for both slab gel electrophoresis and capillary gel electrophoresis. Many experts in the field expect that most parts of human genome can be sequenced within next 3 to 7 years. However, some portions of DNA in human genome which has long repeats and with secondary hairpin structures are still very difficult to be sequenced by conventional gel electrophoresis with Sanger's enzymatic method to produce DNA ladders. The band compression often occur for DNA segments with high GC ratio and/or with secondary structures. PCR process may not faithfully replicate the DNAs which have hair pin structures. Thus, a new and reliable approach to sequence these "difficult" templates is critical for completing the sequencing of the entire human genome. Recently, we tried to couple laser desorption mass spectrometry with Maxam Gilbert chemical degradation method to produce DNA ladders to achieve sequencing of DNA templates with high GC component. DNA templates were first bound with biotin so that DNA ladders produced by the chemical degradation method can be isolated from the solution by magnetic bead streptavidin separation. Then these isolated DNA segments are released from streptavidin and subsequently analyzed by laser desorption mass spectrometry. Since the sequencing by laser desorption mass spectrometry is based on the measurement of molecular weights, band compression is no longer a problem. Since no PCR is required, non-faithful replication by PCR due to the secondary structures or a large number of repeat can be eliminated. 

In addition to the sequencing by Maxam Gilbert's approach, we also used laser desorption mass spectrometry for DNA sequencing for DNA ladders produced by Sanger's method. ss-DNA templates larger than 100 nt were successfully sequenced. ds-DNAs larger than 200 bp were also sequenced. However, mass resolution and detection sensitivity still need more improvement for sequencing longer DNAs. In addition to sequencing DNA with ladders produced by chemical methods, we also developed a technology to produce DNA ladders during the laser desorption process. By controlling the pH value and selecting the right matrices, direct sequencing of short DNA was obtained without the need of the preparation of DNA ladders. Since the sequencing process with this approach is very fast, it can be used to sequence a large number of probes which are often used for diagnosis by hybridization. 

* Research has been supported by DOE/OBER