KR100602903B1 - fluorescent primers for analyzing mitochondrial DNA length heteroplasmy - Google Patents

Abstract

The present invention relates to a method for qualitatively and quantitatively analyzing length heteroplasmy of mitochondrial DNA, including a PCR procedure using fluorescent primers.

According to the present invention, since the fluorescent primer is used as the primer for PCR, the test procedure can be performed quickly and easily, and it is easier to comprehensively grasp the existence of the generated length variants, the components, and the composition ratio thereof. This method can efficiently analyze the length heterogeneity of mitochondrial DNA by saving the cost and time required in this process, and can be expected to be effective in a wide range of fields such as genetics, medicine, and forensics. have.

Mitochondria, DNA, length heterogeneity, isoforms, homologues, fluorescent primers, PCR, electrophoresis, HV1, HV2

Description

Fluorescent primers for analyzing mitochondrial DNA length heteroplasmy}

1A-1F are electropherogram graphs showing length heterogeneity for HV1 in the human mitochondrial DNA genome.

2A-2H are electroperogram graphs showing length heterogeneity for HV2 in the human mitochondrial DNA genome.

(In the graph, 1 refers to the peak representing the highest point, 2 refers to the second high point, 3 refers to the third high point, and 4 refers to the peak representing the fourth high point, and * indicates the smallest peak).

The present invention relates to a method for analyzing the length heteroplasmy of mitochondrial DNA (mitochondrial DNA).

More specifically, the present invention relates to a novel method for qualitatively and quantitatively analyzing the length heterogeneity of mitochondrial DNA through PCR using fluorescent primers.

In general, mitochondria are known as the major organs responsible for energy production in cells, but are the only organelles that contain DNA among the organs outside the cell's nucleus. Due to its biological characteristics, research has been intensively conducted in various fields requiring DNA analysis, including medical field.

Specifically, the human body contains hundreds to tens of thousands of mitochondria per cell, and each mitochondria also has 2 to 10 copies, more than 100 copies of DNA, so that it is easier to detect genetic variation relative to nuclear DNA. In addition to the advantages that can be useful in the diagnosis and treatment of various diseases, the genetic information of the mitochondrial DNA is inherited from the maternal system because of its genetic information is fully available in the field of genetics and forensics.

Human mitochondrial DNA is a double helical circular DNA consisting of 16,569 bp and is usually present in one type of DNA sequence, but in some cases, it is shown to show length heterogeneity in which two or more length variants exist within one individual. The results turned out.

The length heterogeneity of these mitochondrial DNAs, according to recent research data, particularly at the two hypervariable sites in the control region of the mitochondrial genome, namely the first hypervariable segment (HV1) and the second hypervariable segment (HV2). Due to the specificity of its base sequence in the polycytosine stretches found, many researchers, including the present inventors, have diagnosed in mitochondrial-related diseases and cancers, including cerebral nerve disease, heart disease and muscle disease. In the field of therapeutics or related genetics and forensic science, various approaches have been attempted through the study of the length heterogeneity of mitochondrial DNA.

Therefore, as the study of the length heterogeneity of mitochondrial DNA is newly emerging as the object of medical and genetic research, precise and rapid analysis method is eagerly demanded.

Conventionally, methods that have been used for length heterogeneity analysis of mitochondrial DNA include a method of directly sequencing a nucleotide sequence of a desired site [Reference: J. forensic Sci. 2001 Jul; 46 (4): 862-870] or by amplifying the nucleotide sequence of the target site by PCR method, and then separating the resulting product based on the size, thereby confirming the presence, length, and composition of the length isomer. Method [Reference: Am. J. Hum. Genet. 1995 Aug; 57 (2): 248-256.

However, in the prior art of analyzing the length heterogeneity of mitochondrial DNA by the sequencing method, in the case of the HV1 region, several peaks are suddenly lowered through a portion where the same nucleotide C is repeatedly displayed, that is, C-stretch. Although the length heterogeneity can be confirmed by overlapping, it is impossible to confirm the constituents and the ratio of the length isomer in HV1, and in the case of the HV2 site, from the shape of the T (Thymine) peak in the center of the C-stretch. Although the presence of length heterogeneity and the components and ratios of the length isomers can be confirmed, there is a disadvantage in that low ratio heterogeneity of length is not possible due to noise resulting from poor sequencing quality. .

Therefore, for the length heterogeneity analysis of mitochondrial DNA, it is common to use a typing technique based on PCR. This is a method of analyzing the length heterogeneity of the mitochondrial DNA by amplifying a desired repeat nucleotide sequence in the mitochondrial DNA by a PCR procedure, and then separating and detecting the obtained PCR product. By electrophoresis method using polyacrylamide gel or the like, each is separated according to the size difference of the length isoform components, and then stained with a reagent such as silver nitrate or ethidium bromide (EtBr), or a radioisotope. By incorporating radioisotopes during DNA amplification using primers labeled with, and detecting the radioactivity emitted therefrom, the intensity of the band represented by each length isoform on the support was measured, and the obtained data was obtained. Through to determine the components of the length isomers and their proportions. This method, after PCR and amplification product separation, undergoes a separate staining process to determine the result, and then analyzes the intensity using a machine such as an image analyzer, and when performing radioisotopes, Since the radioactivity detection process is not an easy process, the methods that have been used for the length heterogeneity analysis of mitochondrial DNA in the past are time-consuming and expensive and labor when analyzing a large amount of DNA, and experimental errors occur in this process. It is pointed out as a problem that it is difficult to obtain accurate results.

Therefore, the inventors of the present invention have a problem in the prior art by performing a PCR process using a fluorescent primer while studying a method that can be more easily and easily performed in the test procedure in analyzing the length heterogeneity of mitochondrial DNA. The present invention has been completed by finding out that the above disadvantages which have been made can be solved.

The present invention is to overcome the above-mentioned disadvantages of the prior art, in the method for analyzing the length heterogeneity of the mitochondrial DNA by the process of separating the amplification product after the PCR reaction, faster and easier than the prior art It provides an efficient method for analyzing mitochondrial DNA length heterogeneity in terms of time, cost, and reliability by carrying out a test procedure and making it easier and more accurate to comprehensively understand the presence, composition, and composition ratio of the resulting length isomers. It aims to do it.

The present invention relates to a method for qualitatively and quantitatively analyzing the length heterogeneity of mitochondrial DNA by using fluorescent primers in PCR.

More specifically, the present invention

(1) preparing a DNA sample from a specimen obtained from a human body;

(2) in the presence of one or more primers comprising oligonucleotides or derivatives thereof that complementarily bind to a portion of the DNA sequence comprising a portion suspected of exhibiting length heterogeneity on the mitochondrial DNA and labeled with a fluorescent substance; Amplifying the nucleic acid DNA prepared in step (1) by PCR reaction;

(3) separating the obtained amplification products based on size;

(4) qualitatively and quantitatively analyzing the length heterogeneity of mitochondrial DNA, including detecting the presence, composition, and composition ratio of DNA length isoforms by detecting the fluorescence emitted from each isolated product. It is about a method.

Hereinafter, the invention will be described in more detail with reference to the step-by-step process in relation to the method of the present invention.

The present invention is for analyzing the length heterogeneity of mitochondrial DNA, and more particularly for analyzing the length heterogeneity of mitochondrial DNA comprising one or more sequence sites that are assumed to exhibit length heterogeneity.

Mitochondria of human body contains a large number of DNA having length and sequence heterogeneity, and by studying the pattern of the resulting DNA isoform, individual profiling or typing process can be performed. .

In the present invention, sequences estimated to exhibit length heterogeneity in the human mitochondrial genome generally include repeating sequence of several bp having various lengths in the genome, and reflecting the above-described DNA sequences of various lengths in PCR reaction. Refers to a portion of the nucleotide sequence capable of producing a length isoform, such as the polycytosine portion (C-stretch) and agent present at nucleotides 16184 to 16193 within the first hypervariable region (HV1) in the human mitochondrial DNA genome. 2 hypermutation sites (HV2) that comprise the C-stretch moiety present in nucleotides 303-315.

Length heterogeneity of mitochondrial DNA refers to a state in which multiple lengths of mitochondrial DNA having 5 to 13 or more cytosine sequences are present together in one individual, and various types of length isoforms are generated when amplifying nucleic acids by PCR. I can confirm it.

In the method of the present invention for analyzing the length heterogeneity of mitochondrial DNA, step (1) is a process of preparing a DNA sample from a sample obtained from the human body, for example, tissue, saliva, blood, etc. obtained from the human body.

Then, through step (2), the prepared DNA sample is amplified by a conventional PCR reaction.

In this PCR process, the present invention uses an nucleic acid primer labeled with a fluorescent substance, comprising an oligonucleotide or derivative thereof that complements a portion of a DNA sequence that includes a portion that is presumed to be heterogeneous in length. It is a feature of this invention.

Here, 'an oligonucleotide that complementarily binds to a portion of a DNA sequence comprising a portion suspected of exhibiting length heterogeneity' refers to a sequence of about 200 to 300 bp that includes a repeating unit sequence in which length isomers are expected to be produced. Refers to oligonucleotides of several to tens of bp having base sequences that specifically bind to sequences located in front of, or in front of, some of these sequences to enable amplification of the sites.

At this time, as an example of the sequence portions that are assumed to exhibit length heterogeneity, in the case of length heterogeneity studies on the C-stretch of HV1 and HV2 in the human mitochondrial genome, the following two sets each specifically bind to these portions The forward and reverse primers of can be used:

Primer for HV1

Forward L16144 (5'-TGA CCA CCT GTA GTA CAT AA; SEQ ID NO: 1) and

Reverse H16410 (5′-GAG GAT GGT GGT CAA GGG AC; SEQ ID NO: 2),

Primer for HV2

Forward L155 (5'-TAT TTA TCG CAC CTA CGT TC; SEQ ID NO: 3) and

Reverse H389 (5'-CTG GTT AGG CTG GTG TTA GG; SEQ ID NO: 4).

These primers are known in the art, for example, in Crime Lab Digest. 1995; 22: 3-8] and the like, the efficiency thereof has been demonstrated, and can be obtained by simply synthesizing using an oligonucleotide synthesizer (manufactured by Applied Biosystems) or by customizing the relevant company.

In the present invention, fluorescent materials used for nucleic acid primer labeling include 6-FAM (6-carboxyfluorescein), HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7 -dichlorofluorescein), TET (2 ', 7'-dichloro-6-carboxy-4,7-dichlorofluorescein) and 6-JOE (6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein) From the group consisting of, the appropriate one is selected in consideration of the conditions such as chemical binding to the nucleic acid primer, degree of reactivity in the PCR reaction conditions, price.

Nucleic acid primers of the present invention are labeled with fluorescent materials by linking the fluorescent material to the 5'-terminus by phosphoramidite techniques to the primers (Tetrahedron Lett. 1992; 33: 5033-5036.

In this case, the fluorescent material is attached to one of a pair of primers, that is, a forward primer and a reverse primer, and the DNA sequence region whose length heterogeneity is estimated is 2 or more. Different kinds of fluorescent materials are attached to each primer.

PCR process of step (2) using the obtained fluorescent primer is added to the DNA sample obtained in the step (1) in the usual nucleic acid amplification reaction solution containing the fluorescent primer of the present invention, and then in accordance with the general PCR method Perform.

For example, for mitochondrial DNA comprising HV1 and HV2, nucleic acid amplification reactions including Taq polymerase, buffer, MgCl ₂ , dNTP, etc., in addition to the fluorescent primers for the two sets of HV1 and HV2 prepared as described above. DNA sample was added to the solution, and the whole reaction solution was heat-treated at 95 ° C. for 11 minutes, then denatured at 94 ° C. for 1 minute, nucleic acid-primer binding at 56 ° C. for 1 minute, and at 72 ° C. for 1 minute. The procedure of nucleic acid synthesis (extension) is repeated 25 times in sequence, and finally the post PCR is carried out for 45 minutes at 60 ℃, the conventional PCR process is carried out.

As a result of the reaction, a large amount of DNA synthesized from a primer to which a fluorescent substance is attached as an amplification product is produced. They are obtained in the form of length homologs having the same size or in the form of length isomers having various sizes, depending on the length heterogeneity of the sample.

These amplification products are each separated according to their size by the process of step (3).

In the separation process of step (3) in the method of the present invention, DNA such as agarose gel electrophoresis, polyacrylamide gel eletrophoresis, capillary electrophoresis, etc. All conventional methods can be used to isolate and detect fluorescence, using commercial DNA sequencing analyzers to facilitate this process.

As described above, the DNA amplification products generated through the PCR process of step (2) are separated as one or more DNA bands after a predetermined time separation process due to different movement speeds on the support, depending on their length.

For example, homoplasmic DNA amplification products consisting of nucleic acids of the same length only show a single band as a result of electrophoresis, while heteroplasmic DNA amplification products containing nucleic acids of various lengths are types of isoforms. Depending on, it is separated as several bands. In addition, since the fluorescence intensity of each band is emitted according to the concentration of the length isoform, the length heterogeneity of the mitochondrial DNA can be easily determined through step (4) of the present invention for detecting the fluorescence intensity of the band by position. have.

Isolation and analysis of such PCR amplification products are performed using a variety of commercially available software such as ABI PRISM 310 Genetic Analyzers (Applied Biosystems, Foster City, CA, US) and GeneScan Software 3.1 (Applied Biosystems). Can be easily carried out.

Data analyzing the length heterogeneity of human mitochondrial DNA by the method of the present invention is presented in the form of a graph in FIGS. 1A-1F and 2A-2H.

In the case of DNA having length homology as shown in FIGS. 1A and 2A, a single peak pattern is shown, whereas in the case of DNA having length heterogeneity as in FIGS. 1B to 1F and 2B to 2H, According to the type and concentration of the length isoform, the number and height of the peaks show various types of peak patterns.

Through the DNA peak pattern thus obtained, the presence of length heterogeneity for the desired DNA is determined, and the components and composition ratios of the length isomers are measured, and based on these data, qualitative and quantitative analysis of the length heterogeneity is performed. Can be synthesized.

The method of the present invention will now be described in detail by way of example focusing on analysis of the length heterogeneity of the C-stretch portions of HV1 and HV2 in the human mitochondrial genome. The following examples are provided to specifically illustrate and explain the invention, and should not be construed as limiting the invention thereto.

EXAMPLE

Example 1: Human DNA Sample Preparation

In this example, a study was conducted on the length heterogeneity of their mitochondrial DNA in 100 unrelated individuals.

First, prior to the PCR process, in order to collect DNA samples for length heterogeneity analysis of human mitochondrial DNA, the inside of the human body is scraped with a cotton swab, and then a QIAamp DNA mini kit (Qiagen, Hilden, Germany) is used. DNA was extracted. The method is briefly described as follows.

A cotton swab was put in a tube, PBS buffer and proteinase K and AL buffer were added, and then left at 56 ° C. for 10 minutes. Ethanol was added thereto and mixed, and the solution was placed in a QIAamp spin column and spun in a centrifuge to remove the filtrate. AW1 buffer was added to the column, and the spin column was wiped by rotating in a centrifuge, and then wiped again with AW2 buffer. The spin column was transferred to a new tube and AE buffer or distilled water was added and rotated in a centrifuge to obtain an extracted DNA solution.

Example 2 PCR Amplification Procedure for HV1 and HV2 in Mitochondrial DNA Genomes

Prior to the PCR process, fluorescent primers for HV1 and HV2 were first prepared for this process. Primers are described in Prime Lab Digest. 1995; 22: 3-8], according to the primer base sequences for HV1 and HV2, prepared by the general phosphoramidite method as follows.

Since the oligonucleotide is synthesized in the 3 'to 5' direction, the 5'-terminal DMTr (Dimethoxytrityl), which acts as a protecting group, is removed from the phosphoramidite derivative fixed on the support using an acid. The 3 'phosphoramidite derivative corresponding to the deoxynucleotide of was attached using tetrazol as a coupling agent. The unreacted 5 'end was prevented from participating in the next reaction by acetylation with acetic anhydride, and the phosphite triester group generated in the coupling step was converted to phosphoester through an oxidation reaction. This same reaction was carried out several times to synthesize oligonucleotides of the desired sequence, and in the case of primers to which fluorescent substances were attached, the target 5 'was prepared by using a phosphoramidite derivative to which the desired fluorescent substance, FAM or HEX was attached in the last reaction. A fluorescent substance was attached to the end. The oligonucleotides thus synthesized were separated from the support, removed from various protecting groups, and purified by HPLC (High Performance Liquid Chromatography), followed by two sets of fluorescent primers for PCR, forward primer L16144 (5'-) for HV1. TGA CCA CCT GTA GTA CAT AA) and reverse primer H16410 (5'-FAM-GAG GAT GGT GGT CAA GGG AC) and forward primer L155 for HV2 (5'-TAT TTA TCG CAC CTA CGT TC) and reverse primer H389 ( 5'-HEX-CTG GTT AGG CTG GTG TTA GG).

The above example in 10.0 μl PCR reactions consisting of 1.0 U AmpliTaq Gold Polymerase (Applied Biosystems), 1.0 mM MgCl ₂ and 1.0 μm STR buffer (Promega, Madison, WI, USA) containing 200 μM dNTP each. 0.1 ng of the human DNA sample prepared in 1 and 0.2 μM HV1 forward and reverse primers prepared above and 0.3 μM HV2 forward and reverse primers were added and mixed, followed by heat treatment at 95 ° C. for 11 minutes, followed by 94 ° C. 25 cycles of 1 minute (thermal denaturation) at 56 ° C, 1 minute (nucleic acid-primer coupling reaction), and 72 ° C for 1 minute (nucleic acid synthesis reaction), followed by post-treatment at 60 ° C for 45 minutes Thus, a nucleic acid amplification reaction was performed. This heat treatment process was performed using a PTC-200 DNA engine (MJ Research, Watertown, MA, USA).

Example 3: Isolation of PCR Amplification Products

1 μl of the PCR amplification product obtained as a result of the test of Example 2 and 0.5 μl of GeneScan-500 (ROX) size standard (Applied Biosystems) were added to 20 μl of deionized formamide to denature the nucleic acid, which was then ABI PRISM. Separation was performed by capillary electrophoresis using 310 Genetic Analyzers (Applied Biosystems). In this test, POP6 (Applied Biosystems) was used as a polymer to improve resolution, and GS STR A module was used as an analysis module. The data obtained by electrophoresis were analyzed using GeneScan software 3.1 (Applied Biosystems). At this time, a smooth option was not adopted as an analysis parameter to facilitate detection of small peaks.

As a result of testing the DNA samples prepared from 100 subjects in Example 1 by the method of the present invention, it was possible to obtain a graph of various peak patterns as shown in Figures 1a to 1f and 2a to 2h of the accompanying drawings. .

1A-1F illustrate the peak heterogeneity of human mitochondrial DNA for HV1, and FIGS. 2A-2H illustrate the length heterogeneity for HV2, where 1 represents the peak showing the highest peak, Arabic numerals were written according to the height order. In addition, the same numbers are indicated for the peaks having almost the same height, and * are indicated for the minute peaks.

According to the graph, in the case of mitochondrial DNA having length homology, only one peak is shown as shown in FIGS. 1A and 2A, but in the case of mitochondrial DNA showing two or more types of length isomers, According to the type and each concentration, the results were shown in various patterns having various positions, heights, and number of peaks as shown in FIGS. 1B to 1F and 2B to 2H.

In addition, as a result of analyzing the ratio of the corresponding sample for each peak pattern for HV1 and HV2 based on this result, the results shown in Table 1 and Table 2 were obtained.

type N One 64 * 213 * 15 * 312 * 9 * 321 * 8 * 4123 * 3 * 121 * One Sum 100

type N One 31 * 12 * 27 *One* 21 * 12 16 * 123 * 2 * 213 One ** 212 ** One * 2112 * One Sum 100

According to the table, 36% of the DNA samples were found to exhibit length heterogeneity at the HV1 site and 69% of the samples at the HV2 site.

Therefore, according to the length heterogeneity analysis of human mitochondrial DNA using the present invention, in addition to confirming the presence of the length isomers of the DNA samples of interest, the identification of their components and composition ratio, effective typing on the test samples Typing is possible.

When the length heterogeneity analysis of mitochondrial DNA is performed according to the method of the present invention, since the fluorescent primer is used as a primer required for the PCR process, dyeing with a separate compound or a radioisotope is used after the PCR process. Compared to the technology, the entire test process can be performed quickly and easily. Also, using general analyzers and software used in the technical field, it is easier to confirm the existence of the generated length isomers, their components and composition ratios. In this way, it is possible to comprehensively understand the length and heterogeneity of mitochondrial DNA by saving the cost and time required in the process.

In addition, the study of the length heterogeneity of the effective mitochondrial gene by this new analysis method, along with the development of a method for diagnosing or treating related diseases such as cranial nerve disease and myocardial disease, and identifying the cause of the new disease, based on the individual It is expected to be useful in a wide range of fields such as genetics, medicine, forensics, etc., by identifying individuals by typing.

<110> YONSEI UNIVERSITY <120> A METHOD FOR ANALYZING MITOCHONDRIAL DNA LENGTH HETEROPLASMY BY          USING FLUORESCENT PRIMERS <140> 10-2003-0085153 <141> 2003-11-27 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for analyzing mitochondrial DNA length heteroplasmy <400> 1 tgaccacctg tagtacataa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for analyzing mitochondrial DNA length heteroplasmy <400> 2 gaggatggtg gtcaagggac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for analyzing mitochondrial DNA length heteroplasmy <400> 3 tatttatcgc acctacgttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for analyzing mitochondrial DNA length heteroplasmy <400> 4 ctggttaggc tggtgttagg 20

Claims (10)

delete delete delete delete delete A primer for PCR for analyzing the length heterogeneity of mitochondrial DNA, represented by any one of SEQ ID NOs: 1 to 4. Primer for PCR for analyzing the length heterogeneity of mitochondrial DNA, characterized in that the fluorescent material is attached to the oligonucleotide represented by any one of SEQ ID NO: 1 to 4. The method of claim 7, wherein the phosphor is 6-FAM (6-carboxyfluorescein), HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), TET (2 ' , 7'-dichloro-6-carboxy-4,7-dichlorofluorescein) and 6-JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxy fluorescein) Primer. The primer of claim 7 or 8, wherein 6-FAM (6-carboxyfluorescein) is attached to the oligonucleotide of SEQ ID NO: 2. The method according to claim 7 or 8, wherein HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein) is attached to the oligonucleotide of SEQ ID NO: 4 primer.

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