This invention pertains to apparatus for fluorescence detection in electrophoresis systems, and particularly to such apparatus for automatic sequencing of nucleic acids.
The structural analysis of DNA plays an increasingly important role in modern molecular biology. About 4.times.10.sup.6 bases of DNA have been sequenced since the introduction of the enzymatic, or dideoxy, method of rapid sequencing developed by Sanger and his coworkers (Sanger, et al, Proc. Natn. Acad. Sci. U.S.A. 74,5463-5467 (1977), A. J. H. Smith, Meth. Enzymol. 65,560-580 (1980)) and the chemical method developed by Maxam and Gilbert (S. M. Maxam and W. Gilbert, Meth. Enzymol. 65,499-559 (1980)). Typically, four separate reactions are performed on the particular DNA segment to be analyzed. In the enzymatic method, these reactions produce DNA fragments terminating in either adenosine (A), cytosine (C), quanosine (G), or thymidine (T). In the chemcial method, typically, fragments terminating in G, G+A, C+T, or C are produced. In both cases the four sets of reaction products are electrophoresed in adjacent lanes of a high-resolution polyacrylamide gel. An autoradiographic image of the gel is produced, and the autoradiogram is examined to determine the relative lengths of the DNA fragments generated in each of the four reactions. The DNA sequence is inferred directly from that information.
Both of these techniques are very effective but they are also highly labor-intensive, relatively expensive, and involve the use of radioisotopes; values of approximately three to ten thousand bases sequenced per person-year at a cost of one to five dollars per base are representative. For these reasons and since much DNA remains to be sequenced (there are 3.times.10.sup.9 bases in the human genome alone), there has been much recent activity directed toward development of an automated and non-isotopic method of DNA sequence analysis.
One of the more successful attempts has been carried out by Lloyd Smith and coworkers in the laboratory of Leroy Hood at California Institute of Technology (see Bio/Technology, Vol. 3, May 1985). In that approach, four fluorescent dyes with different colored tags are used instead of radioactive labels. Each color corresponds to a different nucleoside so that if the samples are co-separated electrophoretically, the "ladder" of DNA fragments produced during sequencing is segregated into fluorescent multi-colored rungs, each color corresponding to one of the bases A, G, C, or T. As the length of the column is scanned by a fluorescence sensor, the order of the colored bands corresponds to the specific gene sequence. The specific fluorophores selected by Smith, et al, were fluorescein isothiocyanate (FITC) with an emission peak at 520 nm, 4-chloro-7-nitrobenzo-2-oxa-1-diazole (NBD chloride) emitting at 550 nm, tetramethylrhodamine isothiocyanate (TMRITC) emitting at 580 nm, and Texas Red emitting at 610 nm. These emission peaks make the dyes look green, green-yellow, orange-red, and red, respectively.
The specific method used by Smith, et al, was an adaptation of the dideoxy (enzymatic) method of Sanger, which generally involves cloning the gene of interest in the single-stranded DNA phage M13. A primer sequence complementary to the phase sequence adjacent to the cloned gene is used to initiate a DNA synthesis that copies a portion of the gene. In the scheme devised by the Cal Tech group, a single molecule of fluorescent label is linked to each primer. The cloned genes and primers are then placed in four separate DNA synthesis reaction mixtures, each containing all four nucleosides. A small amount of a dideoxy form of a nucleoside, ddATP, ddCTP, ddGTP, or ddTTP is added to each batch. When a dideoxy triphosphate randomly replaces a conventional nucleoside and is incorporated into the developing DNA strand in the synthesis reaction, the nascent DNA copy immediately stops growing. As a result, all strands in the batch with ddATP terminate at a location where adenosine appears in the sequence. Site-specific stops at the C, G, and T positions occur in the other three reaction batches as well.
To distinguish the four bases, a different fluorescent label is used in each reaction mixture. To achieve that, all DNA copies that end in A are labeled with the green-colored FITC; those terminating in C are labeled with the green-yellow NBD chloride, those terminating in G are labeled with the orange-red TMRITC tag, and copies terminating in T are labeled with Texas Red.
In the Cal Tech automated system, aliquots from all four reaction mixtures are electrophoresed through a single polyacrylamide tube gel that sorts the various length fragments by size. Positioned at the bottom of the electrophoresis gel is an argon ion laser that sequentially illuminates each band as it migrates through the gel. When excited by laser light the fluorophores emit at their characteristic wavelength, and the emissions are detected and identified by a sensor. The sequence of emission colors is converted by the machine into a nucleotide sequence.
Although the Cal Tech group has been able to automate the sequencing process, bringing what used to require four lanes into one lane, and have substantially eliminated problems with mobility differences between bases, significant problems still remain to be solved. First, the detection system design is less than optimal in sensitivity. Second, and more importantly, the apparatus can handle only one column at a time, whereas when using autoradiographs many lanes on a slab gel can be sequenced at the same time.
What is needed is a high throughput, real time, fluorescence detection apparatus that can perform nucleic acid sequencing on many lanes of a gel simultaneously. Furthermore, the apparatus should have a high sensitivity but the detection system should not require frequent attention by trained personnel.