JPH07128332A - Method for determining variation in basic sequence - Google Patents

Abstract

PURPOSE:To enhance the capacity for determining the variation with respect to the base by preparing a DNA fragmental sample having terminal bases of three kinds or less through Sanger reaction, subjecting the sample to electrophoretic analysis, and then comparing an electrophoretic pattern thus obtained with a normal basic sequence. CONSTITUTION:DNA fragmental samples are prepared by Sanger reaction for 1 to 3 kinds of terminal bases among adenine(A) guanine(G), thymine (T), and cytosine(C). The samples are labeled with fluorescent elements or radioisotopes, subjected to electrophoretic analysis, and the electrophoretic pattern is compared with a normal basic sequence. Consequently, it can be determined at which position a base T has varied to a base C and at which position a base C has varied to base T. Since each sample requires a fragmental sample for only one kind of base, manhour and cost are reduced to 1/4 and the processing capacity is enhanced significantly.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for determining mutations in various nucleotide sequences of DNA, such as determining mutations in chromosome nucleotide sequences in the field of clinical medicine.

[0002]

2. Description of the Related Art Attempts have been made to elucidate diseases and abnormalities from the nucleotide sequences of genes and viruses, and for this reason, it is important to investigate mutations in the nucleotide sequences. As a method for determining the mutation in the base sequence, a method for determining the entire base sequence of the sample DNA and a method for estimating the mutation by the PCR-SSCP method are used.

In the method for determining the entire base sequence, four kinds of DNA fragment samples whose terminal bases are A (adenine), G (guanine), T (thymine) or C (cytosine) are prepared by the Sanger method. The base sequence is determined by subjecting them to electrophoretic analysis. For example, DN is detected by the Sanger method using a fluorescent primer chemically bound with a fluorescent substance as a label.
A fragment sample is prepared and electrophoresed in separate migration lanes for each end base, and the migration pattern is read online, or
Alternatively, a radioisotope is used as a label, and the migration pattern is photographically processed to determine the base sequence.

[0004] By comparing the total base sequence determined by electrophoresing four types of DNA fragment samples for each end base with a known normal base sequence, it is determined what number base changed from what to what. can do. For example, if the entire base sequence of the sample is determined as ACGT ... If the normal base sequence of this sample is AGGT ..., it can be seen that the second base G has been mutated to C. In the PCR-SSCP method, the presence / absence of a mutation is estimated by utilizing the difference in migration speed depending on the presence / absence of a mutation.

[0005]

In the method of preparing four kinds of DNA fragment samples having different terminal bases by the Sanger reaction, the number of man-hours for preparing the sample by the Sanger reaction is increased, resulting in high cost. When the base sequence is determined by electrophoresis, the end bases A, G, C, and
Since four lanes must be used for each T, there is a problem that the number of samples that can be run at one time is reduced. P
The CR-SSCP method is a method of estimating a mutation and has a problem in reliability.

The present invention is a method for determining a mutation by determining the base sequence of a sample DNA by electrophoresis and comparing it with a normal base sequence. However, the number of steps for preparing a DNA fragment sample for electrophoresis can be reduced. It is intended to reduce the cost and increase the throughput by reducing the number of lanes required for electrophoresis.

[0007]

In the present invention, with respect to a sample DNA of which a normal base sequence is known, a Sanger reaction is carried out on a DNA fragment sample having terminal bases having 3 or less bases out of 4 bases at the ends. The DNA fragment sample prepared by the above method was compared with the normal base sequence of the electrophoretic pattern obtained by the electrophoretic analysis to determine the mutation related to the base of the type of terminal base for which the sample was prepared, and the data related to the sample. And

Normal nucleotide sequences of chromosomes and viruses have been determined by accumulating data so far. When determining the nucleotide sequence of the chromosomal DNA of an affected person or the DNA of an abnormal virus, there is a causal relationship between the case or abnormality and the mutation site of the nucleotide sequence and the type of mutation. It is obtained by stacking. Even if you do not determine all the base mutations in the sample DNA,
By determining the mutations of 1 to 3 types of bases that are closely related to cases and abnormalities, it is possible to determine the correlation with the cases and abnormalities.

In the present invention, based on such knowledge, 1 to 3 types of DNA fragment samples for each terminal base are prepared by the Sanger reaction. The DNA fragment sample is labeled with fluorescence or a radioisotope and analyzed by electrophoresis to obtain migration patterns for the 1 to 3 types of bases. Mutations relating to those bases are determined by comparing the migration pattern with the normal base sequence.

FIG. 1 shows an example in which a DNA fragment sample was prepared by the Sanger method for four types of bases by the conventional method, and the migration pattern was detected by the online electrophoresis method. A, G, C, and T are displayed by different lines. Focusing on the base T, it is known from the knowledge of clinical data to date that the base T at the position shown by the solid arrow does not mutate, and the base T shown by the broken arrow may mutate. It is known.

FIG. 2 shows the nucleotide sequence (CONSEN) of this DNA which is considered to be normal, and the mutations of five types of samples whose total nucleotide sequences were determined by the conventional electrophoresis method. Although each sample has the same DNA, it shows that the base is mutated at different locations depending on the condition or abnormality, and the location and type of mutation differ depending on the sample.

From FIG. 2, the following three types of samples 28c5, 1
For 08c and 622c7, it is possible to identify which type the sample belongs to by determining only the mutation relating to the base T. For the sample 351c4 in FIG. 2, the mutation for the base A may be determined.
For, it is understood that the mutation for the base C may be determined. Therefore, the samples 28c5, 108c, 62
For 2c7, a DNA fragment sample having a base T at the end may be prepared, and for sample 351c4, a DNA fragment sample having a base A at the end may be prepared.
It can be seen that for c3, a DNA fragment sample having the base C at the end should be prepared.

[0013]

EXAMPLE FIG. 3 shows an example of an apparatus used for electrophoretic analysis of a DNA fragment sample prepared by the Sanger method in the present invention. 2 is a slab-like migration gel, and polyacrylamide gel is used. Both ends of the electrophoretic gel 2 are immersed in the electrode layers 4 and 6, and the electrode layers 4 and 6 contain an electrolytic solution. A migration voltage is applied between the electrode layers 4 and 6 by a migration power supply 8.

A sample loading slot 10 for injecting a sample is provided at one end of the electrophoretic gel 2, and the sample is loaded into each predetermined place of the sample loading slot 10. The sample is labeled with a known method by FITC which is a fluorescent substance, and the base A is added to the end depending on the sample by the Sanger method.
It is a DNA fragment sample treated so that any of G, T, and C comes. FITC is excited by an argon laser with a wavelength of 488 nm and emits fluorescence with a wavelength of 520 nm.
When the migration power source 8 is applied, the sample becomes a migration band 16 and migrates in the migration direction 14 in the migration gel 2 over time to be separated, and reaches the measurement section.

If there is an excitation system for irradiating excitation light from an argon laser 18 which emits a laser beam of 488 nm with a condenser lens 20 and a mirror 21, and a migration band 16 where the excitation light beam hits, The fluorescence emitted from the fluorescent substance of the migration band 16 is collected by the objective lens 22, and the 520 nm interference filter 24 and the condenser lens 26 are collected.
To a photomultiplier tube 28 through the optical fiber bundle 27 and a detection system. In the excitation / detection system including the condenser lens 20, the mirror 21, the objective lens 22, the interference filter 24, the condenser lens 26 and the optical fiber bundle 27, the excitation light beam irradiation position is in a direction orthogonal to the migration direction 14 (scanning direction). 29) A scanning stage 30 is provided that moves mechanically so as to scan the measurement line at regular intervals.

Detection signal of the photomultiplier tube 28 (fluorescence signal)
Is taken into a signal processing microcomputer 31 which is a data processing device through an amplifier and an A / D converter 32. When the excitation light beam irradiates the measurement section on the electrophoretic gel 2, the microcomputer 31 also captures a signal corresponding to the position of the irradiation section as scanning data. In this way, all the fluorescence signals obtained by scanning in the scanning direction are taken into the microcomputer 31 together with the location information. A sample prepared by the Sanger method is loaded into the sample loading slot 10 at the upper end of the slab-like electrophoretic gel 2 for each type of terminal base.

The terminal base thus obtained is T 3
The migration patterns for the various types of samples are shown in FIGS. 4 to 6. By comparing these patterns with a known normal base sequence, it is possible to know at which position the base T was mutated to the base C, and at which position the base C was mutated to the base T. In the embodiment, since the DNA fragment sample needs to be prepared by the Sanger method for only one type of base for each sample, the number of steps and the cost are reduced as compared with the conventional case where the sample is prepared by the Sanger method for four types of bases. Is 1/4. In addition, when performing electrophoresis, the number of migration lanes required is 1/4 as compared with the conventional method, so the number of samples that can be analyzed is quadrupled, and the processing capacity is improved. Although it was judged that the determination of mutation of one type of base was not sufficient, it is sufficient to prepare samples for two or three types of bases and subject each to electrophoresis.

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, three or less kinds of DNA fragment samples for each terminal base may be prepared by the Sanger method for each sample. By comparing the electrophoretic pattern of the sample with a normal nucleotide sequence, the presence or absence of mutation can be clearly discriminated by the presence or absence of a peak. And there is no uncertainty as in the PCR-SSCP method. In the present invention, since it is only necessary to prepare three or less kinds of DNA fragment samples for each end base, the number of steps and costs are smaller than those of the conventional preparation of four kinds of DNA fragment samples by the Sanger method. Further, when performing electrophoresis, the number of migration lanes required is smaller than that in the conventional method, so that the number of samples that can be analyzed is increased and the throughput is improved.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of an electrophoretic pattern for determining the entire base sequence by a conventional method.

FIG. 2 is a diagram showing an example of mutation of a sample of the same type as an example of a normal entire nucleotide sequence.

FIG. 3 is a schematic perspective view showing an example of an electrophoretic device used in the present invention.

FIG. 4 is a diagram showing a migration pattern when a sample having a terminal base T is prepared and electrophoresed with respect to the first sample.

FIG. 5 is a diagram showing a migration pattern when a sample having a terminal base T is prepared and electrophoresed with respect to a second sample.

FIG. 6 is a diagram showing a migration pattern when a sample having a terminal base T is prepared and electrophoresed with respect to a third sample.

Claims (1)

[Claims] 1. A DNA fragment sample for which a normal base sequence is known, is prepared by Sanger reaction, and a DNA fragment sample for each terminal base having 3 or less of four types of bases at the ends is prepared by the Sanger reaction. By comparing the migration pattern obtained by electrophoretic analysis with that of a normal base sequence to determine the mutation relating to the base of the type of the terminal base from which the sample was prepared, and use this as the data regarding the sample.