Name: Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells
Uploaded: 2023-04-21T13:20:00
Description: Watch this Scientific Journal Video about Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells at JoVE.com

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

Kvantificering af telomerlængde ved hjælp af ikke-radioaktiv kemiluminescensdetektion

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.

Forfatter Spotlight: Optimering af ydeevneparametre for TAGGG telomerlængdeanalyse  

Optimering af ydeevneparametre for TAGGG Telomere Length Assay

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  

Southern blotting, hybridisering og kemiluminescensdetektion af genomisk DNA isoleret fra A2780-celler

For at udføre sydlig blotting skal du starte med at skære en nylonmembran i et segment på 10 x 12 centimeter. Lav et hak i samme position som gelen. Aktivér membranen ved at dyppe den i destilleret vand efterfulgt af 20X natriumsaltvandscitrat buffer.Anbring en filterpapirvæge på en dobbelt omvendt bakke, sæt således, at enderne berører bunden af den ydre bakke. Placer gelen over filterpapiret. Placer derefter den aktiverede nylonmembran over gelen, og sørg for, at hakene overlapper hinanden.Fjern eventuelle luftbobler med en glasstang. Placer en to centimeter bunke cellulosefilterpapir på 9,5 x 11,5 centimeter og en anden seks centimeter bunke almindeligt filterpapir. Placer en vægt oven på opsætningen for lige vægtfordeling.Fyld den ydre bakke med 20X natriumsaltvandscitrat buffer, og lad den stå natten over for effektiv overførsel. Efter sydlig overførsel fastgøres det overførte DNA på membranen ved UV-tværbinding på en UV-transilluminator. Vask membranen to gange med 25 ml 2X natriumsaltvandcitratbuffer.For at udføre hybridisering inkuberes membranen i 10 ml af den forvarmede præhybridiseringsbuffer. Omrør forsigtigt membranen manuelt med hyppige intervaller. Derefter inkuberes blottet i 10 ml hybridiseringsbuffer ved 42 grader Celsius i tre timer med forsigtig omrøring.Vask blottet to gange i 25 ml streng buffer i 10 minutter ved stuetemperatur med let omrøring. Vask nu blottet to gange i 25 ml forvarmet streng buffer ved 50 grader Celsius i 15 minutter med forsigtig omrøring. Skyl med 15 ml vaskebuffer i fem minutter med let omrøring ved stuetemperatur.Inkuber membranen i 10 ml frisklavet blokeringsopløsning i 30 minutter ved stuetemperatur med forsigtig omrøring. Anbring det derefter i 10 ml anti-digoxigenin konjugeret med en alkalisk fosfatasearbejdsløsning. Vask blottet to gange i vaskebufferen ved stuetemperatur i 15 minutter.Inkuber nu i 10 ml detektionsbuffer i fem minutter ved stuetemperatur. Når du har fjernet den overskydende buffer, skal du placere membranen på et acetatark med DNA-siden opad. Tilsæt ca. en til 1,5 ml substratopløsning dråbevis på membranen.Placer straks et andet acetatark på det og inkuber i fem minutter ved stuetemperatur. Klem den overskydende substratopløsning ud inden billeddannelse. Brug et geldokumentationsbilleddannelsessystem til at indsamle flere umættede billeder på forskellige tidspunkter til analyse.Efter sydlig blotting og hybridisering var telomerrestriktionsfragmenterne tydeligt synlige, angivet med et udtværing.

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Cancer Research

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 ...