JPH06100586A - Labeled primer in non-ri sequencing - Google Patents

Abstract

PURPOSE:To obtain the subject labeled primer, having a specific number of bases in the distance between two biotins and capable of providing good results even if the transfer efficiency is insufficient by labeling an oligonucleotide with plural biotins. CONSTITUTION:A biotin-phosphoramidite reagent is dissolved in acetonitrile so as to provide 0.1M concentration thereof and the resultant solution is then mixed with an oligonucleotide solution to carry out reaction according to the beta-cyanoethyl phosphoramidite method. Thereby, the oligonucleotide is labeled with plural biotins to afford the objective labeled primer, having at least 5 bases of the distance between the two biotins and useful for the non- radioisotopic (non-RI) sequencing. Furthermore, this labeled primer is detected by binding streptavidin to the biotins, then reacting the streptoavidin with a biotinylated alkaline phosphatase, finally labeling the alkaline phosphatase and detecting the resultant labeled substance with a coloring substrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a labeled primer in a method for determining a nucleotide sequence of DNA (hereinafter abbreviated as non-RI sequencing) in which a radioisotope (hereinafter abbreviated as RI) is not labeled. More specifically, it relates to a biotin-labeled primer in a method for determining the base sequence of DNA labeled with biotin.

[0002]

2. Description of the Related Art Conventionally, as a concrete method of DNA sequencing, there are two basic methods, Maxam-Gilbert method (Pro. Natl. Acad. Sci., USA vol.74, p.56).
0, 1977) and the Sanger method (Pro. Natl. Acad. Sci.,
USA vol.74, p.5463, 1977), and RI is mainly used as a marker. These DNA sequencing methods generally consist of the following steps. 1) Selection of DNA clone from sequencing library 2) Base-specific reaction of selected DNA clone 3) Size fractionation of DNA reaction product by polyacrylamide gel electrophoresis 4) Polyacrylamide gel electrophoresis Detection of patterns of DNA bands separated after or during electrophoresis 5) Decoding of DNA bands separated as sequences of bases, A, G, C and T

In 1983, a fully automatic DNA sequencer was developed, and sequencing without using RI as a label has been put into practical use, and non-RI sequencing and automatic sequencing have been realized at the same time. In this automatic sequencer, since the automatically corrected data is output as processed data, the analysis accuracy depends on the software of the sequencer as compared with the conventional RI in which raw data is recorded on X-ray film. Has a high drawback.

Sequencing which does not use RI as a label includes sequencing using a chromogenic substance as an enzyme substrate (Analytical Biochemistry vo.164, p.514-52).
0, 1987), or sequencing using luminescent substances (Nucleic Acids Research vol.17, p.5113-5123)
Have been reported. These methods are called manual type non-RI sequencing. Compared to RI sequencing, non-RI sequencing has a problem of requiring many steps for detecting DNA. In particular, the step of transferring DNA from the separated support to another support in step 4) in the above-mentioned step greatly influences the sequencing result. In this step, the transcription amount of the separated DNA is proportional to the transcription time, but complete transcription is impossible. For this reason, devices and instruments for transferring DNA in a shorter time have been developed.
However, it has not been possible to obtain a sufficient transfer amount in a short time. Further, whether or not the transfer amount is sufficient cannot be confirmed until the final result is obtained, and in the worst case, it is necessary to start over from the first step.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a labeled primer which gives good non-RI sequencing results in non-RI sequencing using biotin as a label even when the transcription efficiency is insufficient. That is.

[0006]

In order to achieve the above object, the present inventors set the number of molecules of biotin, which is a non-RI labeling substance, per sequencing primer to be at least 3 and effectively act for detection. It was found that the distance between the biotins to be used was at least 5 bases, which enabled an increase in the detection sensitivity of short-chain DNA and an increase in the non-RI sequencing even with a low transcription efficiency.

That is, the present invention is a labeled primer for non-RI sequencing, characterized in that the oligonucleotide is labeled with a plurality of biotins and the distance between the two biotins is at least 5 bases.

The labeled primer in the present invention is an oligonucleotide having a base number of about 15 to 50, and it is preferable to bind at least three biotin molecules as a label. The number of bases between biotins is at least 5. 5
If the number is less than the number, there is a drawback that the sensitivity is not increased due to steric hindrance of the enzyme or protein. An example of the labeled primer of the present invention is shown in the sequence listing. As a method for binding biotin to the primer in the present invention, a biotin-phosphoamidite reagent can be used to obtain a primer into which a plurality of biotins have been introduced by the β-cyanoethylphosphoamidite method.

As a method for detecting the labeled primer of the present invention, biotin is labeled with streptavidin, and finally labeled with biotinylated alkaline phosphatase, and a chromogenic substrate such as BCIP (5-Bromo-4-chloro-3-) is used. indolyl-
phosphate) / NBT (Nitro blue tetrazolium) or chemiluminescent substrate such as PPD (4-Methoxy-4 (3-phosphate)
phenyl) spiro [1,2-dioxetane 3,2'-adamantane] disodiu
m salt) to detect.

The non-RI sequencing method using a labeled primer in the present invention comprises the following steps. 1) Selection of DNA clone from sequencing library It is confirmed before sequencing that the DNA fragment is correctly incorporated into the sequencing library. Select colonies or plaques in a medium containing 100 mM IPTG and 2% X-gal, and after culturing, extract DNA to confirm the size and restriction enzyme map. 2) Base-specific reaction of selected DNA clones Sequenase Ver2.0 Sequencing on the obtained DNA
Base-specific reaction is performed by the deoxy method using kit (TOYOBO). 3) Size fractionation of DNA reaction product by polyacrylamide gel electrophoresis 6% polyacrylamide gel electrophoresis is performed at a constant power of about 30 W for about 2 hours. 4) Detection of DNA band pattern separated after polyacrylamide gel electrophoresis or during electrophoresis After completion of electrophoresis, the DNA in the gel was transferred onto a nylon membrane by capillary blotting. Seque
nsing higy Chemiluminescent DNA Sequencing kit (TOY
OBO) is used to enzymatically label the DNA on the membrane. 5) Decoding of DNA bands separated as sequences of bases, A, G, C and T The X-ray film is exposed to chemiluminescence generated by the action of PPD (Lumigen) on the enzyme-labeled DNA to obtain a DNA band.

A conventional method for increasing the sensitivity by increasing the number of labeling substances per molecule of DNA is a method for detecting DNA or protein by the DNA probe method (Addendum.
Anal. Biochem., Vol.137, p.266, 1984, and Proc.
Nat. Sci., USA vol.78, p.6633, 1981) has been reported. However, the number of bases of the probes used in these probe methods is about 500 to 7,000, and if the size of the DNA that can be labeled as in non-RI sequencing is about 17 to about 50 bases, it is arbitrarily labeled. The above DNA probe method for increasing substances cannot be applied. For that reason, the non-RI sequencing label requires three-step labeling starting from biotin with streptavidin and biotinylated alkaline phosphatase. The sensitivity does not increase unless streptavidin or biotinylated alkaline phosphatase binds to one molecule of biotin. In particular, it is expected that the shorter the biotin labeling interval is, the more the binding of streptavidin and biotinylated alariphosphatase is inhibited. The molecular weight of nucleic acid is about 500, while the molecular weight of alkaline phosphatase is about 10
Therefore, it is presumed that the space where two biotin molecules can bind is not necessarily the space where two molecules of streptavidin and biotinylated alariphosphatase cannot bind.

[0012]

EXAMPLES The present invention will be described below with reference to examples. Example 1 Using a biotin reagent prepared by dissolving "Biotin-ON" phosphoramidite reagent (manufactured by Toyobo Co., Ltd.) in acetonitrile to a concentration of 0.1 M, 5 kinds of DNAs shown in the following chemical formula 1 (SEQ ID NO: 1 in the sequence listing) were used. 5 DNAs described in 5) are automatically DN
It was synthesized by an A synthesizer (Applied Bio Systems 381A).

[0013]

[Chemical 1] (In the formula, X represents a biotinylated amidite.) The biotinylated amidite is shown in Chemical formula 2.

[0014]

[Chemical 2] After treating the synthesized DNA with ammonia at 55 ° C. overnight,
Purified by reverse-phase column high performance liquid chromatography, G-
After desalting with 25 columns, each was 0.1pmole / ul
TE buffer (10mM Tris-HCl, pH8.0, 1mM
It was dissolved in EDTA). Next, each of them was diluted in 5 steps and spotted on the hybridization membrane (Hybond N and nylon membrane (manufactured by Amersham Co.)) by 1 ul each. Place the spotted nylon membrane in the hybridization bag and insert the blocking buffer (10 mM
Phosphate buffer, 5% SDS (correct writing), 125 ml NaCl) 2 ml
Was added, the mixture was shaken for 5 minutes, and then discharged. The mixture was shaken twice with 2 ml of 10-fold diluted blocking buffer for 10 minutes each and then discharged. Next, blocking buffer 2 should be 0.5 ug / ml.
Biotinylated alariphosphatase diluted with ml was added, shaken for 5 minutes, and then discharged. Furthermore, Tris buffer (Tr
It was shaken twice with is-HCl, pH 9.2, 1 mM MgCl ₂ , 10 mM NaCl) for 10 minutes each, and then discharged. Then 0.34 mg / ml luminescent substrate,
PPD (manufactured by Lumigen) was added, shaken for 5 minutes, and then discharged.

The X-ray film was brought into contact with the nylon membrane in the hybridization bag and exposed for 10 minutes to detect the DNA on the film. as a result,
In 15 bases, DNA (3) introduced with 3 biotins at a rate of 1 was detected up to 0.05 pmple. D having 10 bases and 3 biotins introduced at a ratio of 1
With respect to NA (4) and 7 bases, a total of 3 biotin-introduced DNA (5) DNAs were detected up to 0.01 pmole. Up to 1 pmole, the DNA (1) having one introduced at the 5'end and the DNA (2) having three consecutive introductions at the 5'end, which have been conventionally used for non-RI sequencing, were detected. Comparison of detection sensitivity by spot test
Shown in.

Example 2 1) Selection of DNA Clone from Sequencing Library Whether or not the DNA fragment is correctly incorporated into the sequencing library is confirmed before sequencing. Colonies or plaques were selected in a medium containing 100 mM IPTG and 2% X-gal, and DNA was extracted after culturing to confirm the size and restriction enzyme map. 2) Base-specific reaction of selected DNA clones Sequenase Ver2.0 Sequencing on the obtained DNA
A base-specific reaction was performed by the deoxy method using the kit (TOYOBO). 3) Size Fractionation of DNA Reaction Product by Polyacrylamide Gel Electrophoresis 6% polyacrylamide gel electrophoresis was performed at a constant power of about 30 W for about 2 hours. 4) Detection of DNA band pattern separated after polyacrylamide gel electrophoresis or during electrophoresis After completion of electrophoresis, the DNA in the gel was transferred onto a nylon membrane by capillary blotting. Seque
nsing higy Chemiluminescent DNA Sequencing kit (TOY
The DNA on the membrane was enzymatically labeled with OBO). 5) Decoding of DNA bands separated as sequences of bases, A, G, C, and T. X-ray film was exposed by chemiluminescence generated by the action of PPD (Lumigen) on enzyme-labeled DNA to obtain a DNA band. .

[0017]

INDUSTRIAL APPLICABILITY In the present invention, by setting the space into which biotin is introduced to at least 5 bases, it becomes possible to label a primer that can be sequenced with a plurality of biotins and increase the sensitivity. Especially 1 in 15 bases,
Maximum (highest) by introducing a total of 3 biotins
And the sequencing was possible even with a transcription amount of DNA which was 1/5 or less of the normal level. That is, in the present invention, it was possible to detect even a smaller amount of DNA by using biotin as a label, and good results were obtained even in non-RI sequencing even with a transcription amount of 1/5 of the conventional amount.

[0018]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: Characteristic symbol: modified base Location: 1 Method of characterizing: P Other information: Nucleotide sequence bound with biotin NGTTTTCCCA GTCACGAC 18

SEQ ID NO: 2 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: Characteristic symbol: modified base Location: 1..3 Method of characterizing: P Other information: Nucleotide sequence bound with biotin NNNGTTTTCC CAGTCACGAC 20

SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features: Characteristic symbol: modified base Location: 1,16,31 Method of characterizing: P Other information: Nucleotide sequence bound with biotin NGCTGCAAGG CGATTNAGTT GGGTAACGCC NGGGTTTTCC CAGTAACGAC 50

SEQ ID NO: 4 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: Characteristic symbol: modified base Location: 1,11,21 Method by which the characteristics were determined: P Other information: Nucleotide sequence bound with biotin NGGTAACGCC NGGGTTTTCC NAGTCACGAC 30

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: Characteristic symbol: modified base Location: 1,8,15 Method of characterizing: P Other information: Nucleotide sequence bound with biotin NAGGGTTNTC CCAGNCACGA C 21

[0023]

[Brief description of drawings]

FIG. 1 shows a comparison of detection sensitivities by a spot test in Example 1.

Claims (1)

[Claims] 1. A labeled primer for non-RI sequencing, wherein the oligonucleotide is labeled with a plurality of biotins, and the distance between the two biotins is at least 5 bases.