1. Field of the Invention
The invention concerns a method and a device for an evanescence-based multiplex sequencing of nucleic acid molecules immobilized on a support.
2. Description of the Prior Art
The sequencing of the human genome consisting of about 3×109 bases or of the genome of other organisms as well as the determination and comparison of individual sequence variants requires the provision of sequencing methods that are rapid and can also be used routinely and inexpensively. Although major attempts have been made to accelerate conventional sequencing methods such as the enzymatic chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74 (1977) 5463) especially by automation (Adams et al. Automated DNA Sequencing and Analysis (1994), New York, Academic Press), at present no more than 2000 bases per day can be determined with a sequencer.
New approaches for overcoming the limitations of conventional sequencing methods have been developed in the last few years which include sequencing by scanning-tunnel microscopy (Lindsay and Phillip, Gen. Anal. Tech. Appl. 8 (1991), 8-13), by highly parallelized capillary electrophoresis (Huang et al., Anal. Chem. 64 (1992), 2149-2154; Kambara and Takahashi, Nature 361 (1993), 565-566), by oligonucleotide hybridization (Drmanac et al., Genomics 4 (1989), 114-128; Khrapko et al., FEBS Let. 256 (1989), 118-122; Maskos and Southern, Nucleic Acids Res. 20 (1992), 1675-1678 and 1679-1684) and by matrix-assisted laser desorption/ionization mass spectroscopy (Hillenkamp et al., Anal. Chem. 63 (1991), 1193A-1203A).
Another method is single molecule sequencing (Dörre et al., Bioimaging 5 (1997), 139-152) in which nucleic acids are sequenced by progressive enzymatic degradation of fluorescent-labelled single-stranded DNA molecules and detection of the sequentially released monomer molecules in a microstructure channel. The advantage of this method is that only a single molecule of the target nucleic acid is sufficient to carry out a sequence determination.
Although considerable advances have been made by using the above-mentioned methods, there is a major need for further improvements. Hence the object of the present invention was to provide a method for sequencing nucleic acids which represents a further improvement over the prior art and which allows a parallel determination of individual nucleic acid molecules in a multiplex format.
A multiplex sequencing method is proposed in PCT/EP01/07462 in which nucleic acid molecules that carry several fluorescent marker groups are provided in an immobilized form on a support and the base sequence of several nucleic acid molecules is determined simultaneously on the basis of the time-dependent change in the fluorescence of the nucleic acid molecules or/and of the cleaved nucleotide building blocks caused by the cleavage of nucleotide building blocks.