DNA Sequencing approaches

We could also show that lymphoblastoid cell lines established from periphetal blood samples from French centenarians showed a significantly higher poly(ADP-ribosyl)ation capacity than control cell iines. The specific en me activity (i.e., cellular PARP-1 activity divided by the amount of cellular PARP-1 protein) was a more powerful parameter than total activity. In a DNA sequencing approach, we identified four new polymorphisms in the human PARP-1 coding region, but none of them was associated with human longevity in our sample. This once again hints to the importance of accessory factors for the r ulation of PARP-1 activity. 

Pentoney, Jr., S. L., Konrad, K. D., and Kaye, W., A single-fluor approach to DNA sequence determination using high performance capillary electrophoresis, Electrophoresis, 13, 467, 1992. 

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

Gene network identification Identify prevalent expression patterns (gene clusters) and then identify DNA sequence patterns This approach has been used to find regulatory elements in yeast. It will soon be used to discover coregulated genes in other organisms (including humans). 

There are, however, many approaches to DNA sequencing, and even some recent instruments such as the sequencer made by LI-COR [28] do not use a CCD. The latter uses instead a very-low-noise silicon avalanche photodetector to excite near infrared-emitting dyes. The source is a laser diode emitting at 785 nm. It is a compact system that can be mounted on a focusing stage with confocal optics and it is meant for small laboratories that do not have HTS requirements. 

Figure 3.11 A simplified overview of the approaches adopted to both chemical and enzyme-based DNA sequencing. Refer to text for details...

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