The video details the procedure for southern blotting, a technique used to transfer DNA from a gel to a nylon membrane. It covers membrane preparation, transfer, hybridization, and detection of DNA fragments. The process culminates in imaging the results, showcasing visible telomere restriction fragments on the membrane.

southern blotting, hybridization, and chemiluminescence detection of genomic dna isolated from a2780 cells

使用非放射性化学发光检测定量端粒长度

Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3&#39; overhang is unable to be replicated by DNA polymerase1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence3. Short telomeres also cause a host of other problems, such as mitochondria dysfunction4,5 and cell dysfunction.
DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year6, resulting in cellular senescence after a certain number of divisions6. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length7. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method8. Because of this, it is not implemented while quantifying telomere length in most studies.
Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.
TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2&#8242;-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

TAGGG端粒长度测定性能参数优化

The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technology is currently used to advance research in the field of telomere length biology are nanopore sequencing and more recently CRISPR Cas based measurement.Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.Telomere restriction fragment analysis or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biologic by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on on each other.

Watch this Scientific Journal Video about Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells at JoVE.com

Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells  

从 A2780 细胞中分离的基因组 DNA 的南方印迹、杂交和化学发光检测

要进行南方印迹，首先将尼龙膜切成 10 x 12 厘米大小的片段。在与凝胶相同的位置做一个缺口。通过将膜浸入蒸馏水中来激活膜，然后使用20X盐水柠檬酸钠缓冲液。将滤纸芯放在双倒置托盘上，设置使两端接触外托盘的底部。将凝胶放在滤纸上。然后将活化的尼龙膜放在凝胶上，确保凹口重叠。用玻璃棒去除任何气泡。放置一堆两厘米的 9.5 x 11.5 厘米大小的纤维素滤纸，以及另外一堆 6 厘米的普通滤纸。将砝码放在设置顶部以实现均匀的砝码分布。用20X盐水柠檬酸钠缓冲液填充外托盘，并放置过夜以进行有效转移。南移后，通过在紫外透射仪上的紫外交联将转移的DNA固定在膜上。用25毫升2X盐水柠檬酸钠缓冲液洗涤膜两次。为了进行杂交，将膜在10毫升预热的预杂交缓冲液中孵育。频繁地手动轻轻搅拌膜。然后将印迹膜在42摄氏度的10毫升杂交缓冲液中孵育3小时，轻轻搅拌。在室温下在25毫升严格的缓冲液中洗涤印迹两次10分钟，轻轻搅拌。现在在25毫升预热的严格缓冲液中在50摄氏度下轻轻搅拌15分钟，洗涤印迹两次。用15毫升洗涤缓冲液冲洗五分钟，在室温下轻轻搅拌。将膜在10毫升新鲜制备的封闭溶液中在室温下轻轻搅拌孵育30分钟。然后将其放入10毫升与碱性磷酸酶工作溶液偶联的抗地高辛中。在室温下在洗涤缓冲液中洗涤印迹两次15分钟。现在在室温下在10毫升检测缓冲液中孵育5分钟。去除多余的缓冲液后，将膜放在乙酸盐片上，DNA面朝上。在膜上滴加约1至1.5毫升底物溶液。立即将另一张醋酸盐片放在其上，并在室温下孵育五分钟。成像前挤出多余的底物溶液。使用凝胶记录成像系统，在不同时间点收集多个不饱和图像进行分析。在南方印迹和杂交后，端粒限制性片段清晰可见，由涂片指示。

southern-blotting-hybridization-chemiluminescence-detection-genomic

Research

JoVE Journal

Cancer Research

Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells

487 次观看.  SVKM's NMIMS Deemed-to-be-University. 要进行南方印迹，首先将尼龙膜切成 10 x 12 厘米大小的片段。在与凝胶相同的位置做一个缺口。通过将膜浸入蒸馏水中来激活膜，然后使用20X盐水柠檬酸钠缓冲液。将滤纸芯放在双倒置托盘上，设置使两端接触外托盘的底部。将凝胶放在滤纸上。然后将活化的尼龙膜放在凝胶上，确保凹口重叠。用玻璃棒去除任何气泡。放置一堆两厘米的 9.5 x 11.5 厘米大小的纤维素...

视频: Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells  

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

https://cloudfront.jove.com/CDNSource/teasers/65288.jpg

科研

癌症研究

A2780 Cells Genomic DNA Isolation and Analysis

Begin by adding 500 microliters of the lysis buffer to the previously obtained A2780 cell pellet, and mix gently using a cut tip with a minimum opening diameter of two millimeters. Add 20 micrograms per milliliter of freshly prepared RNase and mix by gentle inversion. Incubate it at 37 degrees Celsius for 30 minutes.At the end of incubation, add proteinase K to a final concentration of 100 micrograms per milliliter and gently invert 10 times. Incubate again at 55 degrees Celsius for one hour inverting the tube every 10 minutes. Pipette 500 microliters of phenol:chloroform:isoamyl alcohol reagent into the tube and gently invert the tube about 20 times.Centrifuge it at 9, 391 G for 15 minutes at room temperature. Pipette the upper aqueous viscous layer into a new tube. Add an equal volume of chloroform and mix by gentle inversion.After centrifuging the tube once, collect the aqueous layer. Add an appropriate amount of five molar sodium chloride in the tube to increase the concentration by 0.2 moles. Add two volumes of 100%ethanol into the tube and invert it gently 20 times.Centrifuge at 15, 871 G for five minutes at room temperature. After discarding the supernatant, add 500 microliters of 70%ethanol. Centrifuge again at the same condition and then discard the supernatant.Once the pellet is air dried, add 50 microliters of sterile nuclease free water and let it rehydrate. Then mix the DNA by pipetting using the cut tip. Use UV spectrophotometry to measure the concentration of DNA.After digesting the DNA, separate it using 0.8%agarose. Submerge the agarose gel in 0.25 molar hydrochloric acid solution for 10 minutes at room temperature with gentle agitation. Then rinse the gel twice with distilled water.To denature the DNA, submerge the gel in a solution of sodium hydroxide and sodium chloride. Then rinse the gel twice with distilled water. To neutralize the DNA as demonstrated, submerge the gel in a solution of 0.5 molar hydrochloric acid and three molar sodium chloride at pH seven.The extractor genomic DNA showed good integrity indicating its use for further downstream processing of telomere restriction fragments.

A2780 细胞基因组 DNA 分离和分析

To perform southern blotting, start by cutting a nylon membrane into a 10 by 12 centimeter sized segment. Make a notch in the same position as the gel. Activate the membrane by dipping it in distilled water, followed by 20X sodium saline citrate buffer.Place a filter paper wick on a double inverted tray, set such that the ends touch the base of the outer tray. Place the gel over the filter paper. Then place the activated nylon membrane over the gel, ensuring that the notches overlap.Remove any air bubbles with a glass rod. Place a two centimeter heap of 9.5 by 11.5 centimeter sized cellulose filter paper, and another six centimeter heap of regular filter paper. Place a weight atop the setup for equal weight distribution.Fill the outer tray with 20X sodium saline citrate buffer, and leave it overnight for efficient transfer. After southern transfer, fix the transferred DNA on the membrane by UV crosslinking on a UV transilluminator. Wash the membrane twice with 25 milliliters of 2X sodium saline citrate buffer.To perform hybridization, incubate the membrane in 10 milliliters of the pre-warmed pre-hybridization buffer. Gently agitate the membrane manually at frequent intervals. Then incubate the blot in 10 milliliters of hybridization buffer at 42 degrees Celsius for three hours with gentle agitation.Wash the blot twice in 25 milliliters of stringent buffer for 10 minutes at room temperature with gentle agitation. Now wash the blot twice in 25 milliliters of pre-warmed stringent buffer at 50 degrees Celsius for 15 minutes with gentle agitation. Rinse with 15 milliliters of wash buffer for five minutes with gentle agitation at room temperature.Incubate the membrane in 10 milliliters of freshly prepared blocking solution for 30 minutes at room temperature with gentle agitation. Then place it in 10 milliliters of anti-digoxigenin conjugated with an alkaline phosphatase working solution. Wash the blot twice in the wash buffer at room temperature for 15 minutes.Now incubate in 10 milliliters of detection buffer for five minutes at room temperature. After removing the excess buffer, place the membrane on an acetate sheet with the DNA side facing up. Add approximately one to 1.5 milliliters of substrate solution dropwise on the membrane.Immediately place another acetate sheet on it and incubate for five minutes at room temperature. Squeeze out the excess substrate solution before imaging. Using a gel documentation imaging system, collect multiple unsaturated images at different time points for analysis.After southern blotting and hybridization, the telomere restriction fragments were clearly visible, indicated by a smear.