Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-16
2001-12-25
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091520, C436S501000, C436S800000, C536S023100, C536S024320
Reexamination Certificate
active
06333156
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determining the base sequence of nucleic acids, and an apparatus for said methods.
2. Background Art
Determining and specifying the base sequence of nucleic acids, particularly DNAs (deoxyribonucleic acids) and RNAs (ribonucleic acids), are important for elucidating genetic information and biological information of animals (human beings), plants, bacteria, viruses and the like.
Representative conventional methods for determining the base sequence of nucleic acids include a method, called “Sanger method” or “chain termination method,” developed by F. Sanger and A. R. Coulson, and a method, called “Maxam-Gilbert method” or “chemical cleavage method,” developed by A. Maxam and W. Gilbert.
The Sanger method will be briefly explained. A DNA fragment to be sequenced is first introduced into a single stranded DNA phage (for example, M13phage) and cloned. This recombinant DNA is used as a template to anneal a primer DNA, and a complementary oligonucleotide fragment is synthesized using a DNA polymerase. In this case, four deoxynucleotide phosphates as substrates and dideoxynucleotide phosphate as a reaction terminator are added to four separate reaction systems. Upon incorporation of dideoxynucleotide into oligonucleotide being synthesized, the synthesis of the oligonucleotide is terminated. As a result, various lengths of DNA fragments ending with dideoxynucleotide are synthesized. The DNA fragments are electrophoresed on a polyacrylamide gel to detect the fragments based on the label possessed by the DNA fragments, thereby determining the base sequence of the target DNA.
Next, the Maxam-Gilbert sequencing method will be briefly explained. The end of DNA to be sequenced is first labeled, and specific one or two bases are chemically cleaved. In this case, reaction conditions are set so that only several sites are nicked in any DNA. Treatment with piperidine permits DNA to be cut at the cleaved base site to provide various lengths of labeled fragments and unlabeled fragments. Only labeled fragments are involved in the sequencing. The larger the distance of the cleaved base from the labeled one end, the larger the size of the fragment. Electrophoresis permits the distance of the labeled one end to the cleaved base to vary according to the electrophoretic mobility. In this method, it is common practice to carry out the above chemical reaction so that four chemical reactions are carried out respectively to cleave only G, only G and A, only T and C, and only C.
On the other hand, in recent years, an attempt has been made to analyze genomes in a large number of organism species including human beings. In this attempt, there is a strong demand for processing of a large quantity of gene information in a short time. This has led to several proposals on the modification of conventional methods to process a large amount of gene information in a short time. For example, multi-capillary electrophoresis using a plurality of capillary columns has been proposed and put to practical use for processing of a large quantity of gene information in a short time. This method is advantageous in that a plurality of analytes can be analyzed. In this method, however, when one analyte is analyzed, four capillaries should be provided respectively for the bases. In order to simultaneously analyze a plurality of analytes, the number of capillaries should be further increased for each analyte. On the other hand, analysis of one analyte has been carried out using one capillary with a different label being used for each base. In this method, however, for simultaneous analysis of a plurality of analytes, the number of capillaries should be simply increased. Most of multi-capillary electrophoresing devices are constructed so that a plurality of capillaries are scanned by a laser beam or the like to read labels. Therefore, increasing the number of capillaries results in increased width to be scanned, leading to a fear of the detection accuracy being lowered.
Therefore, the development of a method for simultaneously analyzing a plurality of analytes in an efficient and accurate manner has still been desired in the art.
So far as the present inventor knows, there is no report that, in simultaneously analyzing a plurality of analytes by sequencing based on the Sanger method or the Maxam-Gilbert method, oligonucleotide fragments are simultaneously analyzed with a different label being used for each analyte.
SUMMARY OF THE INVENTION
The present inventor has now found that, in simultaneously analyzing a plurality of analytes according to the Sanger method or the Maxam-Gilbert method, use of a different label for each analyte and labeling of oligonucleotide fragments, which have been specifically fragmented with respect to four end bases and are derived from an identical analyte, with an identical labeling material can realize simultaneous analysis of a plurality of analytes with very high efficiency.
Accordingly, it is an object of the present invention to provide a method which can simultaneously analyze a plurality of analytes and can determine the base sequence of a large amount of gene information in a short time, and an apparatus for the method.
According to one aspect of the present invention, there is provided a method for determining the base sequence of target nucleic acids contained in a plurality of analytes, said method comprising the steps of:
(a) providing a plurality of nucleic acid analytes containing respective target nucleic acids;
(b) preparing four samples, for each of the plurality of the nucleic acid analytes, containing various lengths of labeled oligonucleotide fragments respectively having sequences identical to or complementary to a part of the target nucleic acid, the oligonucleotide fragments at their end bases having been base-specifically fragmented, the oligonucleotide fragments having been labeled with a different label for each of the nucleic acid analytes;
(c) subjecting the four samples for each of the nucleic acid analytes to a separation method which can simultaneously distinguish oligonucleotide fragments for each type of end bases, independently of the nucleic acid analytes from which the oligonucleotide fragments have been derived, based on a difference in length of one base; and
(d) detecting the separated oligonucleotide fragments based on the labels and analyzing the length of the oligonucleotide fragments one base by one base to determine the base sequence of the target nucleic acids.
The method for determining the base sequence of a target nucleic acid in a plularity of analytes according to the present invention is based on the fact that the information on the length of oligonucleotide is obtained one base by one base. Thus, according to a second aspect of the present invention, there is provided a method for simultaneously analyzing the length of oligonucleotides, necessary for the determination of the final base sequence, one base by one base for a plurality of sample sets, said method comprising the steps of:
providing a plurality of sample sets each consisting of four samples containing various lengths of labeled oligonucleotide fragments, the oligonucleotide fragments at their end bases having been base-specifically fragmented, the oligonucleotide fragments having been labeled with a different label for each of the sample set;
subjecting the four samples for each of the sample sets to a separation method which can simultaneously distinguish oligonucleotide fragments for each type of end bases, independently of the sample sets, based on a difference in length of one base; and
detecting the separated oligonucleotide fragments based on the labels and analyzing the length of the oligonucleotide fragments one base by one base.
According to a third aspect of the present invention, there is provided an apparatus for simultaneously analyzing the length of oligonucleotides one base by one base for a plurality of sample sets, said apparatus comprising:
test sample holding means for holdi
Chakrabarti Arun Kr.
Fredman Jeffrey
Hayashizaki Yoshihide
Wenderoth , Lind & Ponack, L.L.P.
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