WO2003044228A1

WO2003044228A1 - Improved primer discrimination method in allele-specific primer extension

Info

Publication number: WO2003044228A1
Application number: PCT/KR2002/002143
Authority: WO
Inventors: Jun-Hyeong Cho; Kyoung-Hee Kim; Nam Huh
Original assignee: Samsung Electronics Co., Ltd.
Priority date: 2001-11-23
Filing date: 2002-11-16
Publication date: 2003-05-30
Also published as: AU2002353615A1; KR20030042581A

Abstract

An improved primer discrimination method in allele-specific primer extension performed on a DNA chip is provided. The method uses a primer, which is to be immobilized on a surface or a solid support of the DNA chip, including in a 3' portion an allele-specific nucleotide complementary to an allelic variation nucleotide of a target nucleic acid to be assayed and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide. The accuracy in base sequence analysis and mutant genotyping can be markedly improved. The method is effective in detecting a single point mutation as well as insertion and deletion variations.

Description

IMPROVED PRIMER DISCRIMINATION METHOD IN ALLELE-SPECIFIC

PRIMER EXTENSION

Technical Field

The present invention relates to allele-specific primer extension, and more particularly, to a method for improving the discrimination of primers in allele-specific primer extension (ASPE).

Background Art

The rapid progress in the human genome project has greatly increased the need for fast analysis techniques for a huge amount of genetic information useful in genetic disorder diagnosis, treatment, and prevention. For the last several years, there have been remarkable advances in the manufacture and applications of DNA chips, and particularly, in the base analysis related field.

U.S. Patent No. 5,780,233 discloses a method for discriminating a mutant-free normal nucleic acid target from a variant nucleic acid target, which bears a mutation, using a modified oligonucleotide probe. The modified nucleotide probe bears a true mismatch and at least one artificial mismatch, which are separated from one another by three or four nucleotide positions, and provide a difference in the thermal stability of a normal duplex and a variant duplex formed through hybridization, thereby enabling discrimination of the normal target from the variant target. For a more effective discrimination, the true mismatch base is located in the middle of the modified probe. U.S. Patent No. 5,981 ,176 discloses an allele-specific primer and a method for discriminating between two nucleotide sequences bearing a single base mismatch or identifying an allelic variation nucleotide in a target nucleic acid using the allele-specific primer. The allele-specific primer used in the method contains a 5' portion having a sequence complementary to a preselected nucleotide sequence immobilized at a position on a solid support and a 3' portion having a sequence complementary to the sequence ranging from the allelic variation nucleotide to the 3' end of a target nucleic acid.

Pastinen T. et al. disclose the application of a DNA chip in allele-specific primer extension (ASPE). An RNA template is hybridized to an allele-specific primer immobilized on the DNA chip, and the primer is extended by Moloney murine leukemia virus (MMLV) reverse transcriptase. As a result, genotyping of single nucleotide polymorphisms can be achieved (Genome Research, 10(7): 1031-1042).

In the ASPE method, the oligonucleotide primer is designed to have a 3'-end nucleotide complementary to the sequence of an allelic variation site of a DNA template. The oligonucleotide primer is annealed to a 3' portion of the template DNA extending from the allelic variation site to the 3'-end, and the primer extension is identified using a fluorescently labeled dNTP, thereby gynotyping the allele nucleotide. In this method, four different kinds of primers are used, which have different 3'-end bases, A, G, C, and T, so that only one primer capable of perfectly matching the allele nucleotide participates in the extension reaction, but not the remaining three primers.

However, when the above-described ASPE method is practically applied to an allele nucleotide assay, although the 3' end of the primer cannot form a perfectly matched base pair, the 3' end undergoes a conformational change forming a duplex similar to the duplex resulting from hybridization with a target nucleotide sequence, so that the primer can be extended by enzymes. Accordingly, the allele nucleotide sequence cannot be correctly identified, especially when the 3' end of the primer has G-T base pairs that are more susceptible to the extension reaction.

Disclosure of the Invention

Accordingly, the invention provides an oligonucleotide primer for used in allele-specific primer extension (ASPE) that is not extended when it imperfectly matches an allele nucleotide, so that the allele nucleotide sequence can be accurately assayed.

In an aspect, the invention provides an oligonucleotide primer for used in ASPE, comprising in a 3' portion an allele-specific nucleotide complementary to an allelic variation nucleotide of a target nucleic acid to be assayed and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide. The invention also provides a method for increasing discrimination between primers immobilized on a DNA chip in APSE.

In an aspect, the invention provides a method for increasing discrimination between primers in ASPE, the method comprising: preparing a target nucleic acid including an allelic variation nucleotide; synthesizing a primer including in a 3' portion an allele-specific nucleotide complementary to the allelic variation nucleotide of the target nucleic acid and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide and immobilizing the primer on a solid support; hybridizing the prepared target nucleic acid to the primer immobilized on the solid support and extending the primer by additions of labeled dNTPs and a polymerase enzyme; and screening the primer extension reaction products to identify the allelic variation nucleotide of the target nucleic acid.

Brief Description of the Drawings FIG. 1 shows the sequence of normal HNF-1 a exon 2 gene having 14 allelic variations;

FIGS. 2A and 2B show the sequences of primers and target nucleic acids, which perfectly match (PM) or mismatch (MM) at 3' end, used in a conventional allele-specific primer extension (ASPE) method and in an ASPE method according to the present invention, wherein the primers of FIG. 2B used in the present invention includes an artificial mismatch nucleotide;

FIG. 3 comparatively shows the fluorescent intensities of_PM and MM in the conventional ASPE method and the ASPE method according to the present invention using the primers and target nucleic acids shown in FIGS. 2A and 2B; and

FIG. 4 shows the results of ASPE reactions according to the present invention for different durations, in which the HNF-1 exon 2 gene isolated and amplified from the normal human genomic DNA was used as a target nucleic acid.

Best mode for carrying out the Invention

Hereinafter, the present invention will be described in detail. The present invention provides a primer for use in allele-specific primer extension, comprising in a 3' portion an allele-specific nucleotide complementary to an allelic variation nucleotide of a target nucleic acid to be assayed and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide.

Preferably, the allele-specific nucleotide of the primer, which is

^* complementary to the allelic variation nucleotide of the target nucleic acid, is located at one of the second, third, and fourth nucleotides, and more preferably, at the second nucleotide, from the 3' end of the primer. The artificial mismatch nucleotide of the primer is located at one of the first and second nucleotides from the allele-specific nucleotide in the 5'- or 3'-direction, and more preferably, at the first nucleotide site from the allele-specific nucleotide in the 5'-direction.

Any artificial mismatch nucleotide capable of forming a non-Watson-Crick base pair with a counterpart nucleotide of the target nucleic acid can be used. For example, natural bases capable of forming a pu ne-purine base pair or a pyrimidine-pyrimidine base pair can be used for the artificial mismatch nucleotide. Artificial bases, and more preferably, universal bases, can be used for the artificial mismatch nucleotide. Universal bases are artificial bases that maximize base stacking without collapsing the DNA duplex. A suitable example of a universal base is 3-nitropyrrole. This universal base can bind to four nucleotides, A, C, G, and T with equal strength and thus reduces a variation caused by combination of mismatch pairs.

In the method according to the present invention, the target nucleic acid is a single-stranded DNA (ss-DNA), an ss-RNA, or a double-stranded DNA (ds-DNA). These target nucleic acids can be obtained from a genomic DNA through polymerization chain reaction (PCR) or in vitro transcription. In the method according to the present invention, a polymerase for use in the ASPE can be varied according to the kind of the target nucleic acid used.

For example, T4 DNA polymerase, T7 DNA polymerase, or T. acquaticus DNA polymerase can be used when the target nucleic acid is DNA, and a reverse transcriptase derived from retrovirus can be used when the target nucleic acid is RNA.

In the method according to the present invention, any dNTP that can be detected by light absorption, fluorescence, fluorescence polarization, or mass spectrometry measurement can be added as a labeling material for the primer. However, a suitable example of the labeling material includes Cy2-dUTP or Cy3-dCTP emitting fluorescent signals. In this case, the intensity of the fluorescent signals is analized using a laser scanner, and a variation of the target nucleic acid sequence can be identified from the difference in the intensity of the fluorescent signals for the allele-specific primers. The present invention will be described in greater detail with reference to the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the invention.

Example 1 : Manufacture of DNA chip by Spotting An oligonucleotide probe having a sequence as shown in Table 1 below, which corresponds to a primer used in an ASPE reaction in an example to be described later, was added to 100 mM NaHCO₃ solution (pH 9.0), agitated, and left at 37°C for 1 hour and was used as a spotting solution. The spotting solution was spotted on a surface of a glass substrate, which had been treated to expose amine groups, and left in a wet chamber at 37°C for 4 hours. Subsequently, as a process for background noise control, a region of the glass surface to which the spotting solution was not applied was treated to negatively charge the amine groups of the region and thus to prevent a target nucleic acid from adhering to the non-spotting region, and the resulting glass substrate was incubated in a dryer until used.

Fourteen kinds of variations at exon 2 of HNF-1 α (hepatocyte nuclear factor-1 ) gene can be detected using the probes having the sequences shown in Table 1. In Table 1 , the bold letter sequences are complementary to the sequences of the normal exon 2 gene, and the non-bold letter sequences are complementary to the sequences of exon 2 gene having a point mutation. According to the present invention, those probes in Table 1 have an allele-specific nucleotide complementary to the allelic variation nucleotide of the target nucleic acid at the second nucleotide site from their 3' end and an artificial mismatch nucleotide next to the allele-specific nucleotide complementary to the allelic variation nucleotide in 5' direction.

FIG. 1 shows the sequence of normal HNF-1 exon 2 gene having 14 allelic variations (SEQ ID NO. 1 ). In FIG. 1 , the 14 allelic variation sites are expressed with bold letters, and the underlined sequences correspond to sense and anti-sense primers used in the following example 3.

Table 1

Example 2: Isolation of Genomic DNA

Genomic DNA was isolated using a DNA extraction kit (QAIGEN, USA). 10 mL buffer solution (1.28 M sucrose, 20 mM MgCI₂, 4% Triton X-100, 40 mM Tris-HCI, pH 7.5) and 30 mL distilled water were added into 10 ml of a human blood sample, and left on ice for 10 minutes, and centrifuged at 4000 rpm for 15 minutes. After the supernatant was removed, the pellet was re-suspended in the buffer solution and centrifuged, twice, to isolate blood cells. 5 mL buffer solution (800 mM guanidine-HCI, 30 mM EDTA, 5% Tween-20, 0.5% Triton X-100, 300 mM Tris-CI, pH 8.0) was added to isolated blood cells and re-suspended by Vortex mixer. 200 μL Protease K solution (10 mg/mL) was added into the suspension and reacted in a 50°C-water bath for 1 hour to completely disrupt the blood cells. The disrupted cell solution was centrifuged in a Genomic-tip 100/G filter at 4000 rpm, followed by addition of a buffer solution (1.25M NaCI, 15% isopropanol, 50 mM Tris-CI, pH 8.5) to recover the genomic DNA. Isopropanol was added into the recovered genomic DNA solution, mixed thoroughly, and centrifuged at 4000 rpm for 15 minutes. The supernatant was removed, and the pellet was washed twice with 70% ethanol and centrifuged at 4000 rpm. The resulting pellet was dried and completely dissolved in 10 mM Tris-HCI (pH 8) to obtain a purified genomic DNA.

The concentration of the purified DNA was measured using a spectrophotometer. The concentration of the purified DNA was adjusted to 50 ng/μL to allow more efficient polymerase chain reaction (PCR) in the following example.

Example 3: Amplification of exon 2 of HNF-1 gene by PCR

To amplify the exon 2 fragment of human HNF-1 gene in the genomic DNA isolated in example 2, PCR was performed in a 50 μL reaction volume as follows.

Using 50 ng of the human genomic DNA, 2.5 units of thermo-stable DNA polymerase, 200 μM each dNTP (dATP, dTTP, dGTP, dCTP), 50 mM Tris-HCI (pH 8.3), 40 mM KCI, 1.5 mM MgCI₂, 25 pmol sense primer

(5'-taatacgactcactatagggCGAAGATGGTCAAGTCCTA CCT-3'; SEQ ID NO. 30), and 25 pmol antisense primer (5'-GCCACCTC TCGCTGCTTGC; SEQ ID NO. 31 ), 35 cycles of PCR, each cycle including denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 45 seconds, were carried out, with a pre-denaturation at 94°C for 5 minutes and a last-extension at 72°C for 5 minutes. The sense primer used contained the bactariophase T7 promoter sequence at 5' end, as indicated by small letters.

The amplified PCR product (175 bp) was used to generate labelled single-stranded RNAs through in vitro transcription in the following example 4.

Example 4: Preparation of single-stranded RNA through in vitro transcription

In vitro transcription was carried out at 37°C for 3 hours in a 20 μL reaction volume containing 350 ng of the PCR products in example 3, 40 mM Tris-HCI (pH 7.5), 6 mM MgCI₂, 2 mM spermidine, 10 mM NaCI, 2 mM DTT, 1 mM each ATP, CTP, GTP, UTP, and T7 RNA polymerase (Promega, USA). After completion of the reaction, the reaction product was purified using a QIAquick Nucleotide Removal kit (QAIGEN, USA) by finally adding 10 mM Tris-HCI (pH 8.0). As a result, pure single-stranded RNAs were obtained.

Example 5: Primer extension and sensitivity measurement

10 μL of the single-stranded RNA solution in example 4, 10 mM DTT, 3 mM MgCI₂, 75 mM KCI, 200 μM dATP, 200 μM dGTP, 10 μM Cy3-dUTP, 10 μM Cy3-dCTP, and 200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase were mixed thoroughly to_a total volume of 30 μL, and the mixture was dropped onto the DNA chip manufactured in example 1 , covered with a cover glass, and hybridized at 37°C overnight. The HNF-1 exon 2 probes (primers) immobilized on the chip were hybridized with the single-stranded RNA and simultaneously extended using the RNA as a template by the MMLV reverse transcriptase. Next, the chip was washed with 6X SSPET solution (0.9M NaCI, 60 mM

NaH₂PO₄, 6 mM EDTA (pH 8.0), 0005% (v/v) Triton X-100) for 5 minutes and then with 3X SSPET (0.45M NaCI, 30 mM NaH₂PO₄, 3 mM EDTA (pH 8.0), 0.005% (v/v) Triton X-100).

Sensitivity of the primers in the extension reaction was measured by scanning fluorescent signals from Cy3 using a ScanArray Scanner (GSI Lunonics) on a 10-μm-pixel resolution.

FIGS. 2A and 2B show the sequences of primers and target nucleic acids, which perfectly match (PM) or mismatch (MM) at 3' end, used in a conventional ASPE method and in the ASPE method according to the present invention, respectively. As shown in FIG. 2B, the primers according to the present invention include an artificial mismatch base.

FIG. 3 comparatively shows the degree by which PM and MM are discriminated from one anther in the conventional ASPE method and the ASPE method according to the present invention using the primers and target nucleic acids shown in FIGS. 2A and 2B. The target nucleic acid used had the sequence of 5'-TCGTTGGTCGAAACGGAC-3' and a variation at the sixth base "G" from the 5' end (SEQ ID NO. 32). Four spots for each of the perfect-match and mismatch probes were spotted. The spot diameter was 170±5 μm, and the spot interval was 375 μm. In the conventional method, PM was a primer (probe) immobilized on the

DNA chip and having the sequence of 5'-GTCCGTTTCGACC-3' (SEQ ID NO. 33, the base "C" at the 3' end corresponds to the variation nucleotide site of the target nucleic acid), MM1 was a primer having the same sequence as SEQ ID NO. 33 except for the base "G" at the 3' end, MM2 was a primer having the same sequence as SEQ ID NO. 33 except for the base "A" at the 3' end, and MM3 was a primer having the same sequence as SEQ ID NO. 33 except for the base "T" at the 3' end. In the method according to the present invention, PM was a primer (probe) immobilized on the DNA chip and having the sequence of 5'-GTCCGTTTCGTCC-3' (SEQ ID NO. 34, the second base "C" from the 3' end corresponds to the variation nucleotide site of the target nucleic acid, and the third base "T" from the 3' end is an artificial mismatch base), MM1 was a primer having the same sequence as SEQ ID NO. 34 except for the second base "G" from the 3' end, MM2 was a primer having the same sequence as SEQ ID NO. 34 except for the second base "A" from the 3' end, and MM3 was a primer having the same sequence as SEQ ID NO. 34 except for the second base "T" from the 3' end.

As shown in FIG. 3, PM and MMs in the conventional method had no great differentiation in fluorescence intensity. However, PM and MM2 in the method according to the present invention had a fluorescent intensity of 8414 and 1322, respectively, indicating that PM and MMs can be sensitively differentiated in the present invention.

FIG. 4 shows the results of scanning the DNA chip after ASPE for 30 minutes, 1 hour, and 2 hours according to the present invention. Table 2 below shows the numerical values of the fluorescent intensity of PM and MMs in FIG. 4. The results of FIG. 4 were obtained using the DNA chip on which primers capable of genotyping two allelic variations at adjacent sites were spotted side by side. Probes (variation Nos. 1 through 14) in Table 1 above were sequentially spotted from the left to the right, starting from the uppermost line to the lowermost line.

Table 2

As shown in FIG. 4 and Table 2, the intensity of the fluorescent signals and an intensity ratio of PM (l_PM) to MM (l_MM) varied according to the duration of the primer extension reaction. According to the present invention, the sequence of the normal target nucleic acid was identified with 100 % accuracy. When the ratio of l_PM to l_MM was greater than 1 , the allele nucleotide was determined to be PM. When the ratio of l_PM to l_MM was less than 1 , the allelic base was determined to be MM. The primers according to the present invention can be practically applied to gynotyping target nucleotides having several variation sites. Also, the result of gynotyping using the primers according to the present invention is accurate.

Industrial Applicability

As described above, the present invention provides primers including an artificial mismatch nucleotide in a 3' portion, which can be used for allele-specific primer extension by being immobilized on a glass surface of a DNA chip. The accuracy in base sequence analysis and genotyping can be markedly improved using the primers according to the present invention. The primers according to the present invention can be effectively used in detecting a single point mutation as well as insertion and deletion variations.

Claims

What is claimed is:

1. ^~A primer for use in allele-specific primer extension, comprising in a 3' portion an allele-specific nucleotide complementary to an allelic variation nucleotide of a target nucleic acid to be assayed and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide.

2. The primer of claim 1 , wherein the allele-specific nucleotide is located at one of the second, third, and fourth nucleotides from the 3' end of the primer, and the artificial mismatch nucleotide is located at one of the first and second nucleotide from the allele-specific nucleotide in the 5' or 3' direction.

3. The primer of claim 2, wherein the allele-specific nucleotide is located at the second nucleotide from the 3' end of the primer, and the artificial mismatch nucleotide is located at the first nucleotide from the allele-specific nucleotide in the 5' direction.

4. The primer of claim 1 , wherein the artificial mismatch nucleotide forms a non-Watson-Crick base pair with a counterpart nucleotide of the target nucleic acid.

5. A method for increasing discrimination between primers in allele-specific primer extension, the method comprising: preparing a target nucleic acid including an allelic variation nucleotide; synthesizing a primer including in a 3' portion an allele-specific nucleotide complementary to the allelic variation nucleotide of the target nucleic acid and at least one artificial mismatch nucleotide adjacent to the allele-specific nucleotide and immobilizing the primer on a solid support; hybridizing the prepared target nucleic acid to the primer immobilized on the solid support and extending the primers by additions of labeled dNTPs and a polymerase enzyme; and screening the primer extension reaction products to identify the allelic variation nucleotide of the target nucleic acid.

6. The method of claim 5, wherein the target nucleotide is a single-stranded DNA, a single-stranded RNA, or a double-stranded DNA.