JPH0296653A - Determination of nucleic acid base sequence and reagent kit therefor - Google Patents

Abstract

PURPOSE:To improve the precision in determination of the sequence of bases of a deoxyribonucleic acid by a method wherein a pigment for coloring which is neutral electrically or charged in plus and different in behavior from a fragment of the nucleic acid in a sample thereof at the time of electrophoresis and does not have a functional power being active on the nucleic acid, is added to a solution of the sample of the nucleic acid. CONSTITUTION:A pigment for coloring which is neutral electrically or charged in plus and different in behavior from a fragment of a deoxyribonucleic acid in a sample thereof at the time of electrophoresis and does not have a functional power being active on the nucleic acid is added to a solution of the sample of the nucleic acid. Nile blue A, pararosaniline, neutral red or the like is used as this pigment. The addition of the pigment is made in such a manner that a formamide solution of the pigment of 0.05 to 0.1% is added, in a quantity of 1/3 to an equal quantity, to the sample for electrophoresis. By this method, an improvement in the precision of the quantity of a collected sample and the prevention of a human mistake at the time of injection of the sample are attained, and an improvement in the precision of determination of the sequence of bases of the nucleic acid can be attained.

Description

DETAILED DESCRIPTION OF THE INVENTION (Uses of the invention) The present invention relates to a method for determining nucleic acid base sequences by gel electrophoresis using fluorescent labels and a reagent kit for sequencing. When a sample containing fluorescently labeled nucleic acid fragments is injected into the sample hole, it is electrically neutral or positively charged, and during electrophoresis, the nuclei in the sample i! A coloring dye that behaves differently from fragments and does not have a functional group active against nucleic acids is added.

(Prior art) As a conventional nucleic acid base sequencing method, deoxyribonucleic II
After labeling one end of I (DNA) with a radioactive isotope such as 32p or 353, it is chemically degraded at a specific base, and the resulting fragments of various lengths are subjected to gel electrophoresis and autoradiography. Maxam-Gilbe can read the base sequence from the arrangement of bands on xm film by
rt) method or one wAD NA i: 'fM Irregular DNA Ajawo binding sequence. This is used as a primer and an enzyme such as DNA polymerase is used to synthesize deoxyribonucleoside triphosphate (dNTP) as a substrate. The inhibitor dideoxynucleoside triphosphate (ddN
DNAs with different 3'-end chain lengths were synthesized using a competitive reaction with TP), separated by gel electrophoresis, and labeled with radioactive isotopes by autoradiography.
Dideoxy (also known as Sanger) is known to determine the base sequence by detecting and comparing the positions of NTPs. In all of these methods, the base sequence is determined by subjecting DNA fragments to gel electrophoresis and reading the electrophoresis pattern.

Labeling with radioactive isotopes is used as a means of reading. In contrast, fluorescent labeling has been proposed as a safe labeling method that does not use radioactive isotopes, and Japanese Patent Application Laid-Open No. 60-242368 discloses that a DNA sample is divided into four parts, each of which has a different excitation wavelength. A method is disclosed in which base sequences are determined by electrophoresis using a single column by labeling with a fluorescent dye.

(Problems to be Solved by the Invention) In the nucleic acid base sequencing method using radioactive isotopes, the purpose is to increase the visibility of the sample when injecting the sample containing DNA fragments into an electrophoresis gel, and to observe the electrophoresis state. Usually, about 0.1% of a dye such as bromophenol glue or xylene cyatool is added to the sample. However, in the case-based nucleic acid base sequencing method, the dyes behave similarly to nucleic acids during electrophoresis, and in order to attenuate the excitation light and absorb the emitted light, thereby hindering detection, the nucleobases using radioactive isotopes are used. The dye used in the sequencing method cannot be used, and a colorless and transparent sample is injected into the sample hole. However, the process of probing a colorless and transparent sample in microliter units with a biraperator or microsyringe, etc., and injecting it into the sample hole of a polyacrylamide gel is repeated several thousand times, and it is difficult to repeatedly inject different samples into the same sample. This can easily lead to mistakes, and even if part or all of the sample leaks out of the sample hole, it cannot be confirmed. Furthermore, although it is essential that no air bubbles be introduced into the sample hole, there are drawbacks such as the difficulty of visually recognizing air bubbles in a colorless and transparent sample.

(Means for Solving the Problems) The present inventors have proposed to prevent human error by increasing the visibility of the sample without affecting the results of electrophoresis, and to visually check the addition state of the sample. As a result of repeated studies with the aim of improving the accuracy of polyacrylamide gel electrophoresis, a method of determining nucleic acid base sequencing by making it possible to confirm This completes the present invention.

In other words, the dye added to the colorless and transparent sample injected into the sample hole of the gel electrophoresis device during nucleic acid base sequencing by pre-determining is electrically neutral or positively charged, and the dye is electrically neutral or positively charged. Nucleic acid fragments inside and 111
111 and have no functional groups active against nucleic acids, and in addition, the addition of 'J) 1 gives the sample sufficient coloring to make it visible;
Further, it is more desirable that the dye has sufficient solubility in water or a mixture of water and formamide. In other words, by adding 15 of the above-mentioned dye to the sample, the accuracy of the sample collection amount is improved and human error during sample injection is prevented.During electrophoresis, only negatively charged nucleic acid fragments enter the gel. The nucleic acid fragments are moved toward the lower electrophoresis chamber, and the fluorescent labels on the nucleic acid fragments are sequentially detected by a fluorescent detector. On the other hand, since coloring dyes are electrically neutral or positively charged, they either remain in the sample hole or migrate to the buffer solution in the upper electrophoresis tank, which interferes with the fluorescence detection of nucleic acid fragments. Never. The pigments used in the present invention include Nile Blue A, Pararosaniline, Neutral Red, Basic Tsuksin, Victoria Blue, Victoria Pure Blue BO, Methyl Violet, Thionin, Brilliant Green, Ethyl Violet, Methylene Blue Ethyl Red, and Indophenol Blue. 5
-amino-2,3-cybidelow 1,4-phthalazinedione (CAS#521-31-3), oxazine 9,
Binasia tool, 1.1', 3,3.3'3゛・\xamethylindotricapodianine.

Examples include Bismarck Brown Y, Bismarck Brown R, Astrazal Blue-3RL, Safranin 0, Crystal Violet, Chrysoidine, New Hooksin, and Basacryl Red (CAS#42373-04.-6), which meet the above requirements. Any dye that satisfies the requirements is not limited to these.

The addition of the dye in the present invention is from 0.05 to 0.1 of the dye.
% formamide solution to the sample for electrophoresis.
Adding an equal amount 1: The goal can be achieved more.

In addition, the dye of the present invention can be used in a conventional test for determining interbase dust.
such as brimer, which is a reagent in the dideoxy method, and enzymes typified by the K1 e new fragment.
, 4 rli-type dNTPs, ddNTPs and dNTs
It can also be combined with a mixture of P and ddNTP to form a base sequencing reagent kit. At this time, the dye may be left in powder form, or may be used as a solution in formamide to be used as a reaction stopper.

Example Examples will be explained below.

Example 1 10 microliters of fluorescein-labeled deoxyribonucleotide 211 body solution to 9 parts distilled water
50 microliters was added to dilute the mixture 100 times, and 100 microliters each was dispensed into two separate containers. To one side, 100 microliters of a 0.05% formamide solution of methyl halide was added, and to the other side, 100 microliters of formamide were added, and the mixture was thoroughly stirred to prepare a sample. Sample hole of polyacrylamide gel (゛) microricks for colored sample and uncolored sample respectively],
It was added to the O point, electrophoresed at 1200 volts, detected by fluorescence, and the peak area was calculated and analyzed. As a result, the standard deviation of the n-color sample is 0.08, while the standard deviation of the n-color sample is 0.21.
Therefore, the coloring of the sample contributed to improving the reproducibility of sample addition.

Actual example 2 Fluorescence? ! 0.1% of basic tsuksin for 5 microliters of DNA fragments prepared according to the usual method (base-specific complementary strand synthesis termination reaction) using identified primers.
Add 2 microliters of formamide solution and heat to 95°C.
As a result of analysis using an automatic DNA sequencer after heat treatment for 1 minute, no abnormal peaks were observed and the fluorescence intensity was sufficient compared to the results obtained by normal operation.

(Effects of the invention) When determining the nucleic acid base sequence by gel electrophoresis using fluorescence detection, adding the dye of the present invention to the sample solution increases the visibility of the sample and improves the accuracy of sample collection amount. This prevents human error during sample injection, thereby making it possible to improve the accuracy of nucleic acid base sequence determination using gel electrophoresis.

Claims (1)

[Claims] 1. In a nucleic acid base sequencing method using gel electrophoresis using a fluorescent label, the deoxyribonucleic acid sample solution is electrically neutral or positively charged, 1. A method for determining a nucleic acid base sequence, which comprises adding a coloring dye that behaves differently from a nucleic acid fragment and does not have a functional group active on nucleic acids. 2. The reagents used in nucleic acid base sequencing by gel electrophoresis using fluorescent labels are electrically neutral or positively charged, and behave differently from the nucleic acid fragments in the sample during electrophoresis. 1. A reagent kit for nucleic acid base sequencing, characterized in that it is combined with a coloring dye having no functional group active on nucleic acids.