JPS61219399A - Determination of base sequence in nucleic acid - Google Patents

Abstract

PURPOSE:Only monostrand part in a specific DNA complex is treated with an enzyme to allow the double-strand part to remain unreacting and the hydrolyzate are subjected to separation depending upon molecular weight whereby the titled sequence is decided simply in a usual laboratory. CONSTITUTION:A replicative form of M13 phage DNA (a') of a cloning vector and human lymphocyte DNA (a) are treated with restriction enzymes, respectively to effect recombination and transduction to obtain a temperature (e) in the reaction for synthesizing the complementary chain to the single strand DNA. Then, 4 kinds of complementary chain corresponding to nucleotides constituting the DNA are synthesized to prepare a temperature DNA-complementary DNA complex (f). Then, a nuclease is allowed to act on only the single strand DNA to allow the double-strand part to remain unreacting (g). The resultant DNA fragment mixture is subjected to electrophoresis is to effect separation depending upon molecular weights, the separated DNAs are dyed with silver to read the image of electrophoretic separation whereby the initial sequence of DNA (b') is decided.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a method for analyzing nucleic acids, and particularly to a method for determining nucleic acid base sequences that enables the preparation of nucleic acid fragments suitable for non-radioactive labeling.

[Background of the invention]

One of the conventional nucleic acid base sequencing methods is the chain terminator method (Cell Engineering, VO/, 1.A11pp,
93-101 (1982)). FIG. 1 shows a schematic diagram of a conventional base sequencing method. In Kenpo, four different types of complementary strand elongation termination reagents (ddA, dd
The product (C) of the complementary strand synthesis reaction (b) containing four types of complementary strand synthesis reactions (e, ddG, ddT) is subjected to denaturation treatment (d) such as adding formamide or heating to obtain deoxyribonucleic acid (hereinafter referred to as DNA). ) are all made into single strands, and each reaction product is electrophoretically separated in a different migration path (
e). Template DNA has a large molecular weight (approximately 250,000 daltons) and therefore remains in the sample injection area without being migrated, whereas complementary strand DNA fragments have a small molecular weight (approximately 350 to 35,000 daltons).
000 daltons), so it migrates a distance corresponding to its molecular weight. As a result, DNAs with different chain lengths for each base
Since the fragments are separated by molecular weight, the base sequence of the DNA can be read by interpreting the fragments in descending order of molecular weight.

Conventionally, complementary strand DNA was activated by adding a substrate radioactively labeled with 32p, 3jS, etc. to the synthesis reaction reagent (A in Figure 1), and the electrophoretic separation band was detected and identified with high sensitivity by autoradiography. (~several Pg/separation zone). Since this operation was a chemical tracer experiment using an unsealed radioactive isotope box, strong restrictions were placed on the disposal of the area where it would be carried out, which was an obstacle to simplifying and speeding up DNA sequencing. In addition, as shown in Figure 1(e) as a bracketed electrophoresis band, some DNA fragments with low molecular weights do not incorporate labeled compounds and therefore do not have radioactivity, so they cannot be detected by autoradiography. .

In order to eliminate the above two drawbacks, it is sufficient to be able to directly detect the DNA migration separation zone with high sensitivity without activating the sample. Non-activated DNA detection methods include silver staining and fluorescent staining using fluorescent dyes such as ethidium bromide.
Both have high sensitivity (1 to 5n) for double-stranded DNA.
g), but it is about an order of magnitude less sensitive for single-stranded DNA, so it cannot be applied when DNA fragments are separated as single-stranded, as in the case of the conventional chain terminator method. There wasn't.

[Purpose of the invention]

The purpose of the present invention is to maximize the ability of the detection method when applying nucleic acid detection methods such as silver staining and fluorescent staining to nucleic acid base sequencing using the chain terminator method. D was separated by electrophoresis as stranded DNA.
The object of the present invention is to provide a method that enables electrophoretic separation of NA fragments as double-stranded DNA.

[Summary of the invention]

Nucleic acid base sequencing method using chain terminator method is
In principle, the base sequence can be determined as long as the relative positional relationship of DNA fragments separated by molecular weight by electrophoresis is clarified by some method, so silver staining is an easy-to-handle alternative to conventional radiation detection methods. It is also applicable to non-radiation direct detection methods such as fluorescent staining.

By the way, in the conventional chain terminator method, all DNA fragments contained in the electrophoretic separation zone are single-stranded. On the other hand, silver staining and fluorescent staining have a sensitivity of 11-1 On/migratory band for double-stranded DNA, but for single-stranded DNA
It is estimated that the sensitivity is one order of magnitude worse for A (Proteins/Nucleic Acids/Enzymes, Vot27.410, p.
pl-3.

(1982)' Biochem, 13iophys,
3es, Commun.

VoZ, 102+pp53-58 (1981)).

Therefore, it is effective to change the detection method to a non-radioactive method and convert single-stranded DNA into double-stranded DNA in order to obtain sufficient staining density and fluorescence intensity. In the present invention, the double-stranded DNA generated during the complementary strand synthesis reaction is electrophoretically separated after being cut from the single-stranded portion of the template DNA, thereby making it possible to detect DNA fragments with high sensitivity using a non-radioactive detection method.

[Embodiments of the invention]

An embodiment of the present invention will be described below with reference to FIG. First, the cloning vector Duplicate Deaf M137AgeD
NA (mplll) (a')'t-restriction enzyme BamHI
After digestion and cleavage with (b'), human lymphocyte-derived DNA (a) was added to this site.
A fragment (b) t-, cloned and recombinant DNA (C
) was created. This was introduced into Escherichia coli (JM103) using the calcium chloride method to obtain a transformed bacterium (d).

The transformed bacteria were cultured in eTY medium 2-, and the recombinant (e) of the infectious type M13 mpH secreted outside the cells was recovered (approximately 10 μg to 2 pmot).

This DNA is single-stranded DNA and serves as a template during complementary strand synthesis reaction.

Prior to the next complementary strand synthesis reaction, first, an oligonucleotide (~10 pmot) that specifically pairs with the upstream region of the cloned DNA (bl) for base sequencing is prepared.
is annealed (Fig. 1(a)).

Next, 4, which corresponds to the four types of nucleotides that make up DNA.
Complementary strand synthesis reaction of seeds is carried out to synthesize complementary strand DNA of various lengths with a complementary strand synthesis terminator attached to the end ((f
) ).

Some of these are double-stranded, the rest are single-stranded IJII DN.
A-- Single complementary strand DNA complex (f), single strand DNA
When treated with one unit of DNA-digesting enzyme 81 nuclease that specifically cleaves only A, it is degraded into low-molecular-weight mononucleotides or dinucleotides as shown in (g). However, the double-stranded portion remains undigested.

DNA fragment mixture treated in this way (total amount 1 μg)
was subjected to 12% polyacrylamide gel electrophoresis with a thickness of 0.5 mm and 7 fl in the migration channel, molecular weight separation was performed, and the DNA was stained with silver staining. As shown in (h), the fragmented template DNA was stained at the beginning. Electrophoretic separation images of double-stranded DNA fragments differing in length by one base pair can be obtained, which can be decoded to determine the base sequence of the inserted DNA (bl).

〔Effect of the invention〕

According to the present invention, a double-stranded DNA fragment mixture can be separated by molecular weight according to the chain length, so that the detection ability of non-radioactive DNA detection methods such as silver staining and fluorescent dye staining can be fully utilized. can. In addition, since the base sequence of DNA can be determined without using unsealed radioactive isotopes such as 32P and "S" as labels for DNA, this can be easily carried out in ordinary laboratory facilities, and at the same time The effect is that there is no risk of radiation damage to people.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of a nucleic acid base sequencing method using a conventional chain terminator method using a radiolabeled compound, and FIG. 2 is a schematic diagram showing the procedure of the improved chain terminator method employing the present invention. He he
rumor IJ
%. 11

Claims (1)

[Claims] 1. In nucleic acid base sequencing by the chain terminator method, only the single-stranded portion of the complex is subdivided into a complex of a template deoxyribonucleic acid and a complementary strand deoxyribonucleic acid generated by a complementary strand synthesis reaction. 2.
A nucleic acid base sequencing method characterized by leaving the full-stranded portion unreacted and separating it by molecular weight. 2. In the nucleic acid base sequencing method according to claim 1, the molecular weight of double-stranded deoxyribonucleic acid is separated by electrophoresis, and deoxyribonucleic acid fragments are included by silver staining or fluorescent staining. A nucleic acid base sequencing method characterized by detecting electrophoretic separation zones.