技术领域technical field
本发明涉及DNA检测技术领域，更具体的说是涉及一种基于电转移和真空转移的端粒DNA长度的检测方法。The invention relates to the technical field of DNA detection, in particular to a method for detecting the length of telomere DNA based on electrotransfer and vacuum transfer.
背景技术Background technique
端粒是存在于真核细胞线状染色体末端的一小段DNA-蛋白质复合体，其可保持染色体的完整性，控制细胞分裂周期；已有研究表明，端粒DNA长度的改变与衰老、肿瘤的发生以及DNA修复具有密切的关联性，因此，端粒DNA长度的测定对于生命科学研究而言具有重要意义。Telomere is a small piece of DNA-protein complex at the end of linear chromosome in eukaryotic cells, which can maintain the integrity of chromosome and control the cell division cycle; studies have shown that the change of telomere DNA length is related to aging, tumor The occurrence and DNA repair are closely related, therefore, the determination of the length of telomeric DNA is of great significance for life science research.
Southern杂交是测定DNA的常见方法，将DNA转移到杂交膜上进行分子杂交，显色后即可检测DNA分子长度。然而，现有的转移方法耗时长、转移效率低、重复性差，对于高分子量的DNA转移效果差，进而影响检测结果；因此，如何提供一种快速、高效、稳定的端粒DNA长度的检测方法成为本领域亟待解决的技术问题。Southern hybridization is a common method for determining DNA. The DNA is transferred to a hybridization membrane for molecular hybridization, and the length of the DNA molecule can be detected after color development. However, the existing transfer method takes a long time, has low transfer efficiency, poor repeatability, and poor transfer effect on high-molecular-weight DNA, which in turn affects the detection results; therefore, how to provide a fast, efficient and stable method for the detection of telomere DNA length Become a technical problem to be solved urgently in this field.
发明内容Contents of the invention
有鉴于此，本发明公开了一种基于电转移和真空转移的端粒DNA长度的检测方法，10min即可完成端粒DNA的转移，并且转移后偏差小，检测结果准确、稳定。In view of this, the present invention discloses a method for detecting the length of telomere DNA based on electrotransfer and vacuum transfer, the transfer of telomere DNA can be completed in 10 minutes, and the deviation after transfer is small, and the detection result is accurate and stable.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
一种端粒DNA转移装置，包括负压盒、正极板、DNA转移组件和负极板；A telomere DNA transfer device, comprising a negative pressure box, a positive plate, a DNA transfer assembly and a negative plate;
负压盒为中空的盒体，顶部开口，侧面或底部设置有抽气口；The negative pressure box is a hollow box with an opening at the top and a suction port on the side or bottom;
正极板、DNA转移组件和负极板由下至上依次安装于开口处；The positive plate, the DNA transfer component and the negative plate are installed in the opening sequentially from bottom to top;
正极板、负极板分别连接外接电源的正、负极，并且正极板和负极板上均分布有透气孔；The positive plate and the negative plate are respectively connected to the positive and negative poles of the external power supply, and there are vent holes distributed on both the positive plate and the negative plate;
DNA转移组件包括滤纸、杂交膜和端粒DNA的电泳凝胶块，滤纸、杂交膜和端粒DNA的电泳凝胶块由下至上依次放置于正极板与负极板之间。The DNA transfer component includes a filter paper, a hybridization membrane and an electrophoresis gel block of telomere DNA, and the filter paper, the hybridization membrane and the electrophoresis gel block of telomere DNA are sequentially placed between the positive plate and the negative plate from bottom to top.
DNA转移组件放置于正极板与负极板之间，通过电场作用可使DNA快速转移到杂交膜上；正极板与负极板上均分布有透气孔，由抽气口处抽真空，即可通过负压促进高分子量端粒DNA的转移，进而缩短转移时间，减少转移偏差。The DNA transfer component is placed between the positive plate and the negative plate, and the DNA can be quickly transferred to the hybridization membrane through the action of the electric field; there are air holes distributed on the positive plate and the negative plate, and the vacuum can be evacuated by the suction port to pass the negative pressure. Promote the transfer of high-molecular-weight telomere DNA, thereby shortening the transfer time and reducing transfer deviation.
正极板、负极板可选用导电材质制成具有透气孔的板体。The positive plate and the negative plate can be made of a conductive material to have a plate body with air holes.
优选地，正极板、负极板为采用石墨烯粉和硅砂粉(80-150目)以(1000：1)-(50：1)比例混合，置于模具中高温烧结成的具有直径30-50μm透气孔的电极板。Preferably, the positive plate and the negative plate are mixed with graphene powder and silica sand powder (80-150 mesh) in a ratio of (1000:1)-(50:1), placed in a mold and sintered at a high temperature with a diameter of 30-50 μm Electrode plate with ventilation holes.
优选地，所述杂交膜为NC膜或尼龙膜。Preferably, the hybridization membrane is an NC membrane or a nylon membrane.
优选地，DNA转移组件还包括硅胶膜，硅胶膜上具有开孔，开孔与端粒DNA的电泳凝胶块上的条带区域相对应；硅胶膜放置于杂交膜与滤纸之间。Preferably, the DNA transfer component further includes a silica gel membrane with openings corresponding to the band regions on the electrophoretic gel block of telomeric DNA; the silica gel membrane is placed between the hybridization membrane and the filter paper.
硅胶膜的设置可保证端粒DNA的电泳凝胶块上的条带区域形成较强的负压压力，进而促进端粒DNA的转移。The setting of the silica gel membrane can ensure that the strip region on the electrophoresis gel block of the telomere DNA forms a strong negative pressure, thereby promoting the transfer of the telomere DNA.
优选地，可在硅胶膜上开设多个开孔，安装时每个开孔对应一块端粒DNA的电泳凝胶块的条带区域，进而同时对多块端粒DNA的电泳凝胶块进行处理。Preferably, a plurality of openings can be opened on the silica gel membrane, and each opening corresponds to the strip area of a piece of telomeric DNA electrophoresis gel block during installation, and then multiple pieces of telomeric DNA electrophoresis gel pieces are processed simultaneously .
优选地，上述端粒DNA转移装置，还包括支撑板，支撑板上均匀分布有多个透气口；支撑板安装于开口处；正极板放置于支撑板上。Preferably, the above-mentioned telomere DNA transfer device further includes a support plate, on which a plurality of air vents are evenly distributed; the support plate is installed at the opening; the positive plate is placed on the support plate.
支撑板用于支撑正极板，避免长期使用后正极板产生形变。The support plate is used to support the positive plate to avoid deformation of the positive plate after long-term use.
一种端粒DNA转移方法，使用上述端粒DNA转移装置，将端粒DNA的电泳凝胶块上的端粒DNA转移到杂交膜上。A method for transferring telomere DNA, using the above-mentioned telomere DNA transfer device to transfer the telomere DNA on the electrophoresis gel block of telomere DNA to a hybridization membrane.
上述端粒DNA转移方法，包括如下步骤：The above-mentioned telomeric DNA transfer method comprises the steps of:
(1)组装端粒DNA转移装置，组装过程中滤纸使用转移液润湿，抽气口连通负压抽气泵；(1) Assemble the telomere DNA transfer device. During the assembly process, the filter paper is wetted with the transfer solution, and the suction port is connected to a negative pressure suction pump;
(2)打开负压抽气泵，接通外接电源，进行端粒DNA转移，转移过程中不断向端粒DNA的电泳凝胶块补加转移液。(2) Turn on the negative pressure air pump, connect the external power supply, carry out the transfer of the telomere DNA, and continuously add the transfer solution to the electrophoresis gel block of the telomere DNA during the transfer process.
优选地，端粒DNA转移过程中真空度0.04Mpa，施加电压为3V/cm。Preferably, the vacuum degree is 0.04Mpa and the applied voltage is 3V/cm during the telomeric DNA transfer process.
优选地，转移液为20×SSC。Preferably, the transfer solution is 20×SSC.
使用上述端粒DNA转移装置或上述端粒DNA转移方法将端粒DNA的电泳凝胶块上的端粒DNA转移到杂交膜上，进行杂交，以检测端粒DNA的长度。Use the above-mentioned telomeric DNA transfer device or the above-mentioned telomeric DNA transfer method to transfer the telomeric DNA on the electrophoretic gel block of the telomeric DNA to a hybridization membrane for hybridization to detect the length of the telomeric DNA.
经由上述的技术方案可知，与现有技术相比，本发明在真空转移的基础上引入电场，使得高分子量的端粒DNA转移速度更快，结果更准确、稳定，且适用不同分子量的端粒DNA(人类3-10kb，小鼠100kb左右)。It can be seen from the above-mentioned technical scheme that compared with the prior art, the present invention introduces an electric field on the basis of vacuum transfer, so that the transfer speed of high-molecular-weight telomere DNA is faster, the result is more accurate and stable, and it is suitable for telomeres with different molecular weights. DNA (human 3-10kb, mouse 100kb or so).
附图说明Description of drawings
图1所示为实施例1端粒DNA转移装置结构示意图；Fig. 1 shows the structural representation of the telomere DNA transfer device in Example 1;
图2所示为负压盒结构示意图；Figure 2 is a schematic diagram of the structure of the negative pressure box;
图3所示为实施例2端粒DNA转移装置结构示意图；Fig. 3 shows the structural representation of the telomere DNA transfer device in Example 2;
图4所示为支撑板结构示意图；Figure 4 shows a schematic diagram of the support plate structure;
图5所示为硅胶膜结构示意图；Figure 5 shows a schematic diagram of the structure of the silica gel membrane;
图6所示为本发明方法与传统方法对比结果；Shown in Fig. 6 is the comparison result of the inventive method and traditional method;
其中，1为传统方法转移10min；2为传统方法转移30min；3为实施例3方法转移10min。Among them, 1 is 10 minutes of transferring by the traditional method; 2 is 30 minutes of transferring by the traditional method; 3 is 10 minutes of transferring by the method of Example 3.
图7所示为本发明方法与传统方法对比结果；Shown in Fig. 7 is the comparison result of the inventive method and traditional method;
其中，左图为传统方法转移30min，右图为实施例3方法转移10min；Wherein, the left figure is the transfer of 30min by the traditional method, and the right figure is the transfer of 10min by the method of Example 3;
M为Marker；1为细胞系293T；2为细胞系HeLa；3为细胞系HCT16；4为细胞系HT080；M is Marker; 1 is cell line 293T; 2 is cell line HeLa; 3 is cell line HCT16; 4 is cell line HT080;
图8所示为本发明方法及传统方法与荧光原位杂交结果的一致性比对；Figure 8 shows the consistency comparison between the method of the present invention and the traditional method and the results of fluorescence in situ hybridization;
图9所示为重复性验证试验结果；Figure 9 shows the repeatability verification test results;
其中，左图1-4为传统方法转移30min，右图1-4为实施例3方法转移10min；Among them, the left figure 1-4 is the transfer of 30 minutes by the traditional method, and the right figure 1-4 is the transfer of the method of Example 3 for 10 minutes;
1为细胞系293T；2为细胞系HeLa；3为细胞系HCT16；4为细胞系HT080；1 is the cell line 293T; 2 is the cell line HeLa; 3 is the cell line HCT16; 4 is the cell line HT080;
图10为不同小鼠组织细胞端粒DNA检测结果；Fig. 10 is the detection result of telomere DNA in different mouse tissue cells;
附图标记：1.负压盒；101.安装槽；102.抽气口；2.正极板；3.DNA转移组件；301.滤纸；302.杂交膜；303.端粒DNA的电泳凝胶块；304.硅胶膜；3041.开孔；4.负极板；5.支撑板；501.透气口。Reference signs: 1. Negative pressure box; 101. Installation groove; 102. Air suction port; 2. Positive plate; 3. DNA transfer assembly; 301. Filter paper; 302. Hybridization membrane; 303. Electrophoresis gel block of telomeric DNA ; 304. Silica gel film; 3041. Open hole; 4. Negative plate; 5. Support plate; 501. Air vent.
具体实施方式Detailed ways
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1Example 1
如图1-2所示，一种端粒DNA转移装置，包括负压盒1、正极板2、DNA转移组件3和负极板4。As shown in FIGS. 1-2 , a telomere DNA transfer device includes a negative pressure box 1 , a positive plate 2 , a DNA transfer assembly 3 and a negative plate 4 .
负压盒1为中空的盒体，顶部开口处设置有安装槽101，侧面设置有抽气口102。The negative pressure box 1 is a hollow box body, a mounting groove 101 is provided at the top opening, and an air suction port 102 is provided at the side.
正极板2放置于安装槽101内，DNA转移组件3和负极板4依次放置于正极板2上，各层之间紧密贴合。The positive plate 2 is placed in the installation groove 101, the DNA transfer assembly 3 and the negative plate 4 are placed on the positive plate 2 in sequence, and the layers are closely attached to each other.
正极板2、负极板4分别连接外接电源的正、负极。正极板2和负极板4制备方法如下：采用石墨烯粉和100目硅砂粉以50:1比例混合，置于模具中高温烧结成具有直径30-50μm透气孔的电极板。The positive plate 2 and the negative plate 4 are respectively connected to the positive and negative poles of an external power supply. The preparation method of the positive plate 2 and the negative plate 4 is as follows: mix graphene powder and 100-mesh silica sand powder at a ratio of 50:1, put them in a mold and sinter at high temperature to form an electrode plate with air holes with a diameter of 30-50 μm.
DNA转移组件3包括滤纸301、杂交膜302和端粒DNA的电泳凝胶块303，滤纸301、杂交膜302和端粒DNA的电泳凝胶块303由下至上依次放置于正极板2与负极板4之间。DNA transfer assembly 3 includes filter paper 301, hybridization membrane 302 and electrophoresis gel block 303 of telomere DNA, and filter paper 301, hybridization membrane 302 and electrophoresis gel block 303 of telomere DNA are placed on the positive plate 2 and the negative plate in sequence from bottom to top between 4.
杂交膜302为尼龙膜。The hybridization membrane 302 is a nylon membrane.
端粒DNA的电泳凝胶块303为样品端粒DNA的琼脂糖凝胶块。The electrophoresis gel block 303 of telomere DNA is an agarose gel block of sample telomere DNA.
实施例2Example 2
如2-5所示，一种端粒DNA转移装置，包括负压盒1、支撑板5、正极板2、DNA转移组件3和负极板4。As shown in 2-5, a telomere DNA transfer device includes a negative pressure box 1 , a support plate 5 , a positive plate 2 , a DNA transfer assembly 3 and a negative plate 4 .
负压盒1为中空的盒体，顶部开口处设置有安装槽101，侧面设置有抽气口102。The negative pressure box 1 is a hollow box body, a mounting groove 101 is provided at the top opening, and an air suction port 102 is provided at the side.
支撑板5为不锈钢网板，其上均匀分布有多个透气口501；支撑板5放置于安装槽101内。The support plate 5 is a stainless steel mesh plate, on which a plurality of air vents 501 are evenly distributed; the support plate 5 is placed in the installation groove 101 .
正极板2、DNA转移组件3和负极板4依次放置于支撑板5上，各层之间紧密贴合。The positive plate 2, the DNA transfer assembly 3 and the negative plate 4 are sequentially placed on the support plate 5, and the layers are closely attached to each other.
正极板2、负极板4分别连接外接电源的正、负极。正极板2和负极板4制备方法如下：采用石墨烯粉和100目硅砂粉以100：1比例混合，置于模具中高温烧结成具有直径30-50μm透气孔的电极板。The positive plate 2 and the negative plate 4 are respectively connected to the positive and negative poles of an external power supply. The preparation method of the positive plate 2 and the negative plate 4 is as follows: mix graphene powder and 100-mesh silica sand powder at a ratio of 100:1, put them in a mold and sinter at high temperature to form an electrode plate with air holes with a diameter of 30-50 μm.
DNA转移组件3包括滤纸301、硅胶膜304、杂交膜302和端粒DNA的电泳凝胶块303，滤纸301、硅胶膜304、杂交膜302和端粒DNA的电泳凝胶块303由下至上依次放置于正极板2与负极板4之间。DNA transfer assembly 3 comprises filter paper 301, silica gel membrane 304, hybridization membrane 302 and electrophoresis gel block 303 of telomere DNA, filter paper 301, silica gel membrane 304, hybridization membrane 302 and electrophoresis gel block 303 of telomere DNA from bottom to top Placed between the positive plate 2 and the negative plate 4.
硅胶膜304中央开设有一开孔3041，开孔3041与端粒DNA的电泳凝胶块上的条带区域相对应，开孔的大小不小于端粒DNA的电泳凝胶块上条带区域的面积。There is an opening 3041 in the center of the silica gel membrane 304, the opening 3041 corresponds to the band area on the electrophoresis gel block of telomeric DNA, and the size of the opening is not smaller than the area of the band area on the electrophoresis gel block of telomeric DNA .
杂交膜302为尼龙膜。The hybridization membrane 302 is a nylon membrane.
端粒DNA的电泳凝胶块303为样品端粒DNA的琼脂糖凝胶块。The electrophoresis gel block 303 of telomere DNA is an agarose gel block of sample telomere DNA.
实施例3端粒DNA长度的检测方法The detection method of embodiment 3 telomeric DNA length
1.端粒的制备(基因组DNA使用Qiagen69506试剂盒提取)1. Preparation of telomeres (genomic DNA was extracted using Qiagen69506 kit)
(1)10cm细胞培养皿中培养细胞，弃细胞培养液，PBS洗2次，胰酶37℃消化3min，收集到15mL离心管中，200g离心3min，弃上清液，PBS清洗细胞2次，转至1.5mL离心管中。(1) Culture cells in a 10cm cell culture dish, discard the cell culture medium, wash twice with PBS, digest with trypsin at 37°C for 3 minutes, collect in a 15mL centrifuge tube, centrifuge at 200g for 3 minutes, discard the supernatant, wash the cells twice with PBS, Transfer to a 1.5mL centrifuge tube.
(2)加入200μL BufferTL，吹打混匀后，加入20μL蛋白酶K，57℃水浴锅中振荡消化30-60min至细胞完全裂解。(2) Add 200 μL of BufferTL, mix by pipetting, then add 20 μL of proteinase K, shake and digest in a 57°C water bath for 30-60 minutes until the cells are completely lysed.
(3)加入220μL BufferBL，漩涡混匀，50℃水浴锅中振荡静置10min。(3) Add 220 μL BufferBL, vortex to mix, shake and stand in a 50°C water bath for 10 minutes.
(4)加入220μL无水乙醇，漩涡混匀。(4) Add 220 μL absolute ethanol, and vortex to mix.
(5)将试剂盒中的DNA吸附柱放在2mL收集管中，将上一步得到的所有液体加至吸附柱中，13000rpm(15871g)离心1min。(5) Put the DNA adsorption column in the kit into a 2mL collection tube, add all the liquid obtained in the previous step to the adsorption column, and centrifuge at 13000rpm (15871g) for 1min.
(6)弃滤液，将吸附柱放回2mL离心管中，向吸附柱中加入BufferKB 500μL，13000rpm(15871g)离心30s。(6) Discard the filtrate, put the adsorption column back into a 2 mL centrifuge tube, add 500 μL of BufferKB to the adsorption column, and centrifuge at 13000 rpm (15871 g) for 30 s.
(7)弃滤液，将吸附柱放回2mL离心管中，加DNAWashingBuffer 650μL，13000rpm(15871g)离心1min。(7) Discard the filtrate, put the adsorption column back into the 2mL centrifuge tube, add 650μL of DNA WashingBuffer, and centrifuge at 13000rpm (15871g) for 1min.
(8)弃滤液，将吸附柱放回2mL离心管中，加DNAWashingBuffer450μL，13000rpm(15871g)离心1min。(8) Discard the filtrate, put the adsorption column back into a 2 mL centrifuge tube, add 450 μL of DNA Washing Buffer, and centrifuge at 13000 rpm (15871 g) for 1 min.
(9)弃滤液，将吸附柱放回2mL离心管中，13000rpm开盖离心2min，去除残留乙醇。(9) Discard the filtrate, put the adsorption column back into a 2 mL centrifuge tube, and centrifuge at 13000 rpm for 2 min to remove residual ethanol.
(10)将吸附柱放入新的1.5mL离心管中，在吸附柱膜中央加100μL预热(70℃)的ElutionBuffer(Real-time PCR用)或无酶水(SouthernBlot和C-circle用)，室温静置1-3min，13000rpm(15871g)离心1min洗脱DNA。(10) Put the adsorption column into a new 1.5mL centrifuge tube, add 100 μL of preheated (70°C) ElutionBuffer (for Real-time PCR) or enzyme-free water (for SouthernBlot and C-circle) to the center of the adsorption column membrane , let stand at room temperature for 1-3min, and centrifuge at 13000rpm (15871g) for 1min to elute the DNA.
(11)取1μg步骤(10)获得的基因组DNA使用HinfI和RsaI进行限制性酶切消化2小时，将基因组DNA消化，并保留端粒DNA。(11) Take 1 μg of the genomic DNA obtained in step (10) and perform restriction digestion with HinfI and RsaI for 2 hours to digest the genomic DNA and retain the telomere DNA.
2.端粒琼脂糖凝胶电泳2. Telomere Agarose Gel Electrophoresis
取500ng端粒DNA加入6×Loading缓冲液，混匀后上样；1-2V/cm，端粒DNA从负极泳向正极；电泳至溴酚蓝指示剂接近凝胶另一端时，停止电泳。Add 500ng of telomere DNA to 6×Loading buffer, mix well and load the sample; 1-2V/cm, telomere DNA is electrophoresed from the negative electrode to the positive electrode; when the bromophenol blue indicator is close to the other end of the gel, stop the electrophoresis.
3.转膜3. Transfer film
将琼脂糖凝胶中的DNA转移到尼龙膜上，形成固相DNA；具体步骤如下：Transfer the DNA in the agarose gel to a nylon membrane to form a solid-phase DNA; the specific steps are as follows:
(1)组装实施例2端粒DNA转移装置：(1) Assembling the telomere DNA transfer device of Example 2:
将支撑板5放置于安装槽101内，正极板2放置于支撑板5上。滤纸301铺设于正极板2上，使用20×SSC转移液润湿滤纸301。然后将硅胶膜304、杂交膜302依次铺设于滤纸301上，取步骤3获得的端粒DNA的电泳凝胶块303，置于杂交膜302上，并使端粒DNA的电泳凝胶块303上的条带区域位于硅胶膜304上开孔3041的位置。将负极板4放置于端粒DNA的电泳凝胶块303上。各层之间紧密贴合。正极板2、负极板4分别连接外接电源的正、负极。抽气口102连通负压抽气泵。The support plate 5 is placed in the installation groove 101 , and the positive electrode plate 2 is placed on the support plate 5 . The filter paper 301 is laid on the positive electrode plate 2, and the filter paper 301 is wetted with 20×SSC transfer liquid. Then the silica gel membrane 304 and the hybridization membrane 302 are successively laid on the filter paper 301, the electrophoresis gel block 303 of the telomeric DNA obtained in step 3 is placed on the hybridization membrane 302, and the electrophoresis gel block 303 of the telomeric DNA is The strip area is located at the position of the opening 3041 on the silicone membrane 304 . The negative plate 4 is placed on the electrophoretic gel block 303 of telomeric DNA. There is a tight fit between the layers. The positive plate 2 and the negative plate 4 are respectively connected to the positive and negative poles of an external power supply. The suction port 102 is connected with a negative pressure suction pump.
(2)打开负压抽气泵，接通外接电源，进行端粒DNA转移，真空度为0.04Mp，施加电压为3V/cm，转移时间10min，转移过程中由负极板4顶部不断向端粒DNA的电泳凝胶块303补加转移液。(2) Turn on the negative pressure pump, connect the external power supply, and transfer the telomere DNA. The vacuum degree is 0.04Mp, the applied voltage is 3V/cm, and the transfer time is 10min. During the transfer process, the telomere DNA is continuously transferred from the top of the negative plate 4 The electrophoresis gel block 303 is supplemented with transfer solution.
4.杂交4. Hybridization
利用端粒探针进行杂交，显色后进行长度检测。利用Telotool软件，将端粒弥散条带均匀划分成20个区域，对每个区域进行亮度统计，根据分子量marker进行换算后统计(JanettNick Fulcher,Jaroslaw Jacak,Karel Riha et al.2014.Telo Tool:anew tool for telomere length measurement from terminal restriction fragmentanalysis with improved probe intensity correction.Nucleic Acid Res,42：21-27)。The telomere probe was used for hybridization, and the length was detected after color development. Using Telotool software, the telomere diffuse band was evenly divided into 20 regions, and the brightness statistics were performed on each region, and the statistics were converted according to the molecular weight marker (Janett Nick Fulcher, Jaroslaw Jacak, Karel Riha et al. 2014. Telo Tool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction. Nucleic Acid Res, 42: 21-27).
实施例4Example 4
使用实施例3方法对人宫颈癌细胞HeLa 1.2.11(美国辛辛那提大学CarolynPrice实验室)端粒DNA(端粒长度18kb)进行检测，并且以传统的真空转移方法替换步骤3进行比较。The method in Example 3 was used to detect the telomere DNA (telomere length 18kb) of human cervical cancer cell HeLa 1.2.11 (Carolyn Price Laboratory, University of Cincinnati, USA), and the traditional vacuum transfer method was used to replace step 3 for comparison.
传统的真空转移方法：将滤纸、尼龙膜和端粒DNA的电泳凝胶块按由下到上的顺序摆放在端粒DNA转移槽中，并除去气泡。打开负压抽气泵，进行抽滤转膜，期间不断在胶面补加转移液20×SSC。转移时间分别为10min、30min。Traditional vacuum transfer method: Place the filter paper, nylon membrane and electrophoretic gel blocks of telomere DNA in the telomere DNA transfer tank in order from bottom to top, and remove air bubbles. Turn on the negative pressure pump to carry out suction filtration and membrane transfer, during which the transfer solution 20×SSC is continuously added to the surface of the rubber. The transfer time was 10min and 30min respectively.
实验结果如图6所示，使用实施例3方法进行端粒DNA转移10min的效果优于传统方法转移30min的效果。The experimental results are shown in FIG. 6 , the effect of transferring telomere DNA for 10 minutes using the method of Example 3 is better than the effect of transferring 30 minutes by the traditional method.
实施例5Example 5
使用实施例3方法及实施例4中传统方法分别对细胞系293T(ACS-4500，端粒长度5.2kb)、HeLa(CCL-2，端粒长度5.4kb)、HCT16(CCL-247EMT，端粒长度7.0kb)、HT080(CRL-12012，端粒长度9kb)的端粒DNA进行检测，结果如图7所示。由于端粒DNA长度的计算时大分子量DNA所占权重较高，因此实施例3方法所产生结果更加接近端粒实际长度。Cell line 293T ( ACS-4500, telomere length 5.2kb), HeLa ( CCL-2, telomere length 5.4kb), HCT16 ( CCL-247EMT, telomere length 7.0kb), HT080 ( CRL-12012, telomere length 9kb) was detected, and the results are shown in FIG. 7 . Since the weight of large molecular weight DNA is higher in the calculation of the length of telomere DNA, the result produced by the method in Example 3 is closer to the actual length of telomere.
进一步地，使用荧光原位杂交方法测定端粒DNA长度(Zijlmans JMJM,MartensUM,Poon SSS,Raap AK,Tanke HJ,et al.(1997)Telomeres in the mouse have largeinter-chromosomal variations in the number of T2AG3 repeats.Proc Natl AcadSci 94:7423–7428.)。如图8所示，实施例3方法与荧光原位杂交结果的一致性较高，偏差小。Further, the length of telomeric DNA was measured by fluorescence in situ hybridization (Zijlmans JMJM, MartensUM, Poon SSS, Raap AK, Tanke HJ, et al. (1997) Telomeres in the mouse have largeinter-chromosomal variations in the number of T2AG3 repeats .Proc Natl AcadSci 94:7423–7428.). As shown in Figure 8, the consistency between the method of Example 3 and the results of fluorescence in situ hybridization is high, and the deviation is small.
进一步地，使用实施例3方法及实施例4中传统方法对4种细胞端粒DNA重复检测3次，结果如图9所示，本发明实施例3方法重复性更佳。Furthermore, the method in Example 3 and the traditional method in Example 4 were used to repeatedly detect the telomere DNA of 4 kinds of cells three times, and the results are shown in FIG. 9 .
实施例6Example 6
使用实施例3方法分别对小鼠尾、小鼠肝脏和小鼠脑细胞端粒DNA进行检测，如图10所示，本发明方法同样适用于大片段端粒DNA。The method of Example 3 was used to detect the telomeric DNA of mouse tail, mouse liver and mouse brain cells respectively, as shown in FIG. 10 , the method of the present invention is also applicable to large fragments of telomeric DNA.
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对上述实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
| Application Number | Priority Date | Filing Date | Title |
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| CN201910828910.1ACN110607221B (en) | 2019-09-03 | 2019-09-03 | Method for detecting telomere DNA length based on electrotransfer and vacuum transfer |
| Application Number | Priority Date | Filing Date | Title |
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| CN201910828910.1ACN110607221B (en) | 2019-09-03 | 2019-09-03 | Method for detecting telomere DNA length based on electrotransfer and vacuum transfer |
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| CN110607221Atrue CN110607221A (en) | 2019-12-24 |
| CN110607221B CN110607221B (en) | 2021-12-28 |
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| CN201910828910.1AActiveCN110607221B (en) | 2019-09-03 | 2019-09-03 | Method for detecting telomere DNA length based on electrotransfer and vacuum transfer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112126580A (en)* | 2020-09-04 | 2020-12-25 | 深圳市龙岗区耳鼻咽喉医院 | Nucleic acid transfer membrane device and nucleic acid transfer membrane method |
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| US5217592A (en)* | 1985-09-26 | 1993-06-08 | Jones Kenneth W | Electrophoresis and vacuum molecular transfer apparatus |
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| CN109439517A (en)* | 2018-12-26 | 2019-03-08 | 天康生物股份有限公司 | Dot hybridization reaction unit and its application and immune blotting detection method |
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| US5217592A (en)* | 1985-09-26 | 1993-06-08 | Jones Kenneth W | Electrophoresis and vacuum molecular transfer apparatus |
| US4911816A (en)* | 1986-02-04 | 1990-03-27 | Oncor, Inc. | Process for conducting electrophoresis and transfer |
| US5269931A (en)* | 1990-09-17 | 1993-12-14 | Gelman Sciences Inc. | Cationic charge modified microporous membranes |
| US5279721A (en)* | 1993-04-22 | 1994-01-18 | Peter Schmid | Apparatus and method for an automated electrophoresis system |
| CN1240831A (en)* | 1998-07-03 | 2000-01-12 | 新疆保利达科工贸有限责任公司 | Nucleic acid pulse hybrid method |
| CN109439517A (en)* | 2018-12-26 | 2019-03-08 | 天康生物股份有限公司 | Dot hybridization reaction unit and its application and immune blotting detection method |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126580A (en)* | 2020-09-04 | 2020-12-25 | 深圳市龙岗区耳鼻咽喉医院 | Nucleic acid transfer membrane device and nucleic acid transfer membrane method |
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| CN110607221B (en) | 2021-12-28 |
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