JPH1066600A - Primer for detecting human cytochrome p450 2c9 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new primer, comprising an oligonucleotide having a specific base sequence and capable of amplifying a human cytochrome P450 2C9 gene without amplifying human cytochrome P450 2C17, 2C18 and 2C19 genes. SOLUTION: This new primer comprises an oligonucleotide having a base sequence represented by the formula and is useful for amplifying a human cytochrome P450 2C9 gene. Since the primer is capable of specifically amplifying only the human cytochrome P450 2C9 gene without amplifying human cytochrome P450 2C17, 2C18 and 2C19 genes, the polymorphism of the human cytochrome P450 2C9 gene can accurately be diagnosed. The primer is obtained by chemically synthesizing an oligonucleotide having a base sequence capable of hybridizing with a 245-267nt region counted from the first base of the 7th exon of the human cytochrome P450 2C9 gene as 1nt with a DNA synthesizer, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human cytochrome P4.
50 2C9 gene amplification primers.

[0002]

[Prior Art] Cytochrome P450 2C9 (hereinafter "CYP2C9")
Are enzymes that catalyze the metabolism of various drugs. Therefore, investigating whether there is an abnormality in the amino acid sequence of CYP2C9 is important for predicting the presence or absence of side effects to various drugs in patients.

[0003] Since about 1987, various researchers
CYP2C9 cDNA and amino acid sequence analysis has been actively performed, and amino acid mutations have been reported. In response to these reports, Wang et al. In 1995 targeted Chinese people in all the places reported so far (exon3 (Arg144-C
ys), exon7 (Tyr358-Cys), exon7 (Ile359-Leu) and exon
8 (Gly417-Asp) mutation) was subjected to gene polymorphism analysis by PCR-RFLP method (Su-Lan Wang et al., Phar
macogenetics (1995) 5, 37-42). As a result, for Chinese, the exon7 (Ile359-Leu) mutation (ie, CYP2C9
Is a mutation in which the isoleucine at position 359 becomes leucine (due to a point mutation in exon 7).

[0004]

The above exon7 (Ile359-Le)
u) When examining the presence or absence of a mutation by the PCR-RFLP method, first, the above-mentioned point mutation in exon 7 (183 nt of the 183 nt with the first base of exon 7 as 1 nt)
It is necessary to amplify the region encompassing the site by PCR, and the specific sequence of the primer for that is also described above.
It is described in Wang et al. However, CYP
There are also enzymes in the family, 2C17, 2C18 and 2C19, whose gene sequence is very similar to 2C9. Using the primers described in the above-mentioned Wang et al.
When the CYP2C9 gene is amplified, not only the CYP2C9 gene,
It was found that the CYP2C17 gene and the CYP2C19 gene were also amplified. If the CYP2C17 gene or the CYP2C19 gene is amplified, these bands will be detected in RFLP, which may lead to erroneous diagnosis. For example, in the RFLP described in the following example, the PCR product
Digest with KpnI and examine the pattern by gel electrophoresis.The amplified CYP2C17 or CYP2C19
Since it is not digested with nI, a band appears at the same position as wild-type CYP2C9. Therefore, even when the sample is homozygous for the mutant type, since a band appears at the position of the wild type, it is misdiagnosed as heterozygous between the wild type and the mutant type.

Accordingly, an object of the present invention is to provide CYP2C17, CYP2
Without amplifying C18 and CYP2C19 genes, CYP2C9
An object of the present invention is to provide a primer capable of specifically amplifying only a gene.

[0006]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention have found that by using a novel primer different from that described in the above literature as a reverse primer, the CYP2C17, CYP2C18 and CYP2C19 genes can be obtained. The present inventors have found that it is possible to specifically amplify only the CYP2C9 gene without amplifying the gene, and completed the present invention.

[0007] That is, the present invention relates to SEQ ID NO: 1
And a primer for amplifying a human cytochrome P450 2C9 gene comprising an oligonucleotide having a base sequence represented by

[0008]

DETAILED DESCRIPTION OF THE INVENTION As described above, the human CYP2 of the present invention
The primer for C9 amplification is composed of an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. This primer uses the first base of the seventh exon of the human CYP2C9 gene as 1 nt (hereinafter, particularly referred to as Unless otherwise noted, "~ nt" refers to the seventh of the human CYP2C9 gene.
(Meaning the number counted as the first base of the exon as 1 nt). It hybridizes to a region of 245 nt to 267 nt.

When the human CYP2C9 gene is amplified using the primer of the present invention, the primer of the present invention is used as a reverse primer (ie, hybridizes with the downstream end of the amplification region). As the primer on the forward side, any primer can be used as long as it hybridizes with a region upstream of 183 nt. For example, the primer described in the above-mentioned Wang et al. (Shown in SEQ ID NO: 2 in the sequence listing) can be preferably used as it is. The forward primer shown in SEQ ID NO: 2 was 163
nt to 182 nt (the 17th base G of this forward primer)
Is originally A corresponding to 239 nt, but has been changed to G to create GGTACC, a cleavage site for KpnI).

[0010] The method of gene amplification itself is well known in the art. A typical example is PCR (polymerase c
The reaction can be easily carried out since a device and a reagent kit for PCR are commercially available. However, the primer of the present invention
In addition, the present invention can be applied to any gene amplification method using primers.

The above-mentioned 183 nt point mutation can be examined by digesting the amplified DNA fragment with an appropriate restriction enzyme and subjecting it to electrophoresis to examine its migration pattern. For example, when the primer described in the above-mentioned Wang et al. Is used as the forward primer, KpnI can be used as the restriction enzyme. In this case, 183 nt is wild type, that is,
In the case of A, since there is no KpnI cleavage site in this portion, the amplified DNA fragment is not cleaved at all, and when separated by electrophoresis, only one band having a size of 105 bp appears. On the other hand, when 183 nt is a mutant type, that is, in the case of C, a cleavage site of KpnI is present, so that the amplified fragment is cleaved between 182 nt and 183 nt. And 85
bp band, a total of two bands appear. Therefore, KpnI
And subjecting it to electrophoresis, it is easy to determine whether 183 nt is A or C.

FIG. 1 shows the method exemplified above (ie, a method of using an oligonucleotide having the sequence of SEQ ID NO: 2 as a forward primer and digesting with KpnI).
1 schematically shows an electrophoresis pattern obtained by the above method. Since somatic cells have chromosomes inherited one by one from their parents, there are two CYP2C9 genes per cell. When the two genes are wild-type homozygous (that is, when both 183 nt are A (indicated by “A / A” in FIG. 1), as shown in FIG. 1, one band of 105 bp is present. On the other hand, in the case of heterozygous wild type and mutant type (indicated by “A / C” in FIG. 1), a single band of 105 bp is generated in the wild type gene and 20 bp in the mutant type gene. And two bands of 85 bp, resulting in a total of three bands.Moreover, in the case of the mutant homozygous (indicated by “C / C” in FIG. 1), two genes are Since both bands produce a band of 20 bp and a band of 85 bp, a total of two bands appear, so that the genotype of each subject can be determined by the above method.

The sample to be subjected to the gene amplification method is as follows:
Any material may be used as long as it contains genomic DNA. For example, DNA extracted from peripheral lymphocytes of a subject by a conventional method can be used as a sample.

The primer of the present invention is commercially available DN
Chemical synthesis can be easily performed using an A synthesizer or the like.

[0015]

The present invention will be described more specifically below with reference to examples.

Example 1, Comparative Example 1 (1) Synthesis of Primer Using a commercially available DNA synthesizer, the oligo DNA primer of the present invention having the base sequence represented by SEQ ID NO: 1 in the Sequence Listing (Example 1, hereinafter This primer was sometimes referred to as "R.K3"). For comparison, 273n
Wang et al., hybridizing at t-293 nt
"Rh" (SEQ ID NO: 3), which is a reverse-side oligo DNA primer described in the above-mentioned reference, was also synthesized (Comparative Example 1). Further, as the oligo DNA primer on the forward side, a primer having the above-mentioned sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as “Fi”) was also synthesized.

(2) Commercially available D from lymphocytes in human blood
DNA was extracted by a conventional method using an NA extraction kit, and this was used as a sample. Using the combination of the primers Fi and R.K3 or the combination of Fi and Rh, PCR was performed using a commercially available PCR kit and the sample DNA as a template. The PCR reaction solution had the composition shown in Table 1 below.

[0018]

[Table 1]

In the PCR, first, pre-denaturation is performed at 94 ° C. for 5 minutes, followed by 30 cycles of denaturation (94 ° C., 30 seconds), annealing (64 ° C., 30 seconds), and extension (72 ° C., 1 minute). This was performed by repeating the reaction twice and finally performing extension at 72 ° C. for 5 minutes.

[0020] The PCR amplification product was transferred to Bluescript SK Vector.
(Trade name, manufactured by APPENDIX) or pCRIIVector (trade name, In
(manufactured by Invitrogen) in a conventional manner. After transforming Escherichia coli by mixing the competent cells and the ligation reaction solution, the cells were cultured overnight in an LB medium. Harvest and pL
Plasmid DNA was prepared by an ordinary method using ASmix Minipreps (trade name, manufactured by TALNET). Dye Terminator Cyc
le Sequencing Kit (PERKIN ELMER) or ^T7 Sequencing K
Using it (trade name, manufactured by Pharmacia Biotech), 15 to 16 clones were sequence-analyzed to find out what the amplified gene was. The results are shown in Table 2 below.
Shown in

[0021]

[Table 2]

As is clear from Table 2, when the primer of the present invention was used, all of the amplified genes were CYP2C9
However, when known primers were used, 25% of the amplified genes were CYP2C17 or CYP2C19. Therefore, by using the primer of the present invention,
It turns out that only CYP2C9 can be specifically amplified.

Example 2 The cycle of PCR was 1 minute for denaturation, 1 minute for annealing,
Except that the elongation is 2 minutes, the same as in Example 1,
PCR was performed using DNA in healthy human peripheral blood lymphocytes as a sample.

The PCR amplification product was digested with KpnI and subjected to 4% agarose gel electrophoresis (100 V, 30 minutes).
The gel was taken out and the band pattern was confirmed under an ultraviolet light. The genotype of each individual was determined from the band pattern. As a result, 209 wild-type homozygotes (A / A)
5.9%) and 9 heterozygous (A / C) (4.1)
%), And the number of mutant homozygous (C / C) was 0 (0%).

[0025]

According to the present invention, a primer capable of specifically amplifying only the CYP2C9 gene has been provided for the first time. When the primer of the present invention is used, only the CYP2C9 gene can be specifically amplified, so that a polymorphism in the CYP2C9 gene can be correctly diagnosed.

[0026]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 23 Sequence type: Nucleic acid Sequence ACAAACTTAC CTTGGGAATG AGA 23

SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid sequence TGCACGAGGT CCAGAGGTAC 20

SEQ ID NO: 3 Sequence length: 21 Sequence type: nucleic acid sequence AAACATGGAG TTGCAGTGTA G 21

[Brief description of the drawings]

FIG. 1 shows an electrophoresis pattern and CY when PCR-RFLP is performed using primers according to one embodiment of the present invention.
It is a figure which shows the relationship with the type of P2C9 gene typically.

Claims (1)

[Claims] 1. A human cytochrome comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 1 in the sequence listing
Primer for P450 2C9 gene amplification.