Plant Biotech 6 (2017)Apunte Inglés
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Southern blot analyssis
The aim of the Southern blot is to detect the presence of a specific sequence. For its procedure,
an average of 5 days is needed.
It has 3 main steps, DNA isolation, the southern blot itself and the Hybridization with the specific probe.
Plant DNA isolation We need to break the cells to free the DNA. The most efficient method is to use liquid Nitrogen and ground the samples to fine powder with a mortar. Then, we homogenize the powder with an Extraction Buffer.
By this procedure, the low temperature keeps all the structures unaltered, and the molecules won’t be oxidized.
The Extraction Buffer has components with a very determinate aim: - 500 mM NaCl: the cells break due to high molarity 1,5% SDS: the detergent interferes with the stability of the membranes 100 mM Tris-HCl: has high buffering properties, maintaining the pH at a specific value.
In this case pH 8, because DNA is more stable at this determinate pH 50 mM EDTA: absorbs the bivalent ions, it’s an ion-chelating agent (Calcium, Magnesium,...). Nucleases need those bivalent ions, so they lose its function. And DNA is protected.
The next step is the Phenol-Chloroform extraction.
By this procedure we can remove almost all the proteins present in the aqueous mixture. Those proteins bind to the DNA won’t be removed.
At the start proteins and DNA are in an aqueous mixture. But is phenol is added and mixed, proteins change their conformation by flipping the less polar residues outside. Then, by centrifugation, the two phases are separated. And the DNA is kept at the upper aqueous part, and most of the proteins are kept in the Phenolic part.
Then, DNA is mixed with alcohol, in which DNA is not soluble. DNA will precipitate forming white droplets or filaments, condensed DNA molecules.
We could do all these procedures with a Handmade protocol or with a Kit.
The kit is more expensive and the yield on DNA recovered is lower. The Handmade protocol permits an “unlimited” quantity of plant tissue.
When analysing the quantity of DNA and RNA in the sample we use the new generation of Spectrophotometers (Nanodrop) which gives us the OD. At 260nm we measure the RNA and DNA. At 280 nm the proteins. By the ration between them we can determine the yield of the extraction. The ratio of 260nm/280nm is an estimate of DNA purity. A value between 1,6 and 2,1 tells us that the DNA is GOOD.
Plant Biotech Tema 6 Héctor Escribano Southern blot analyssis Southern blot It’s a technique that enables us to identify the presence of a certain protein with a certain probe.
By a Genomic DNA analysis, we can study the presence of specific genes and how genes are organized in the genome, looking at the number of copies and/or the transgene integration sites.
First, you have to digest the DNA, using a restriction enzyme. The choice will vary on the sequence you want to detect. That choice is crucial. Depending on the enzyme used we will be able to study the number of copies of a gene of interest, the integration of a transgene in the host genome and/or its integration patterns, and the gene methylation (epigenetic analysis).
Second, you need to separate all the double strand DNA in order to run it in an Agarose gel. For the analysis 10 to 25 micrograms of DNA are needed.
For the digestion, we need some units of enzyme. A unit is defined as the amount of enzyme needed to digest 1 microgram of bacterial virus lambda DNA in 1 hour in a 50 uL reaction. Just in case, a 10 times concentration is used.
Never ever go over the 10% of the final volume of the total reaction with enzyme volume.
Because the enzyme is stored in glycerol (enables it to survive at -20 ºC), that inhibits the digestion if present in sufficient quantities.
BSA is also used, to reduce toxicity as it absorbs wastes.
Once the DNA is digested, an efficiency check is needed. To do that we run a minigel of Agarose with an aliquot of the digested DNA. Once you check, you do the big gel. A small percentage of Agarose is used to facilitate migration along the gel and to facilitate the movement of DNA from the gel to the Solid support.
In the minigel you can use high voltage to do a fast separation (45 minutes), but in the real gel, you need to make a fine separation so a lower voltage is used (20-30V – 16 hours).
The gel needs to be prepared for the transference (the blot).
Depurination makes a partial hydrolysis of the DNA by adding HCl, without destroying it completely. 5 minutes at 0,25 M HCl. Not all the fragments are broken at the same points, so probe will bind to some of them.
Denaturation separates the strands of DNA. Using high concentration of NaCl. After, the DNA must be stabilized again with Tris to keep its optimum pH.
By capillary action and water movement, the DNA is carried along the saline solution upwards and is transferred to the Nitrocellulose/Nylon layer.
Then, the DNA must be fixed to the membrane. It can be done by temperature or by UV light.
The UV light causes the creation of a covalent bond between chemical groups of the membrane and Thymine residues.
Plant Biotech Tema 6 Héctor Escribano Southern blot analyssis If the gene of interest is 1kb long, a probe of 500 bp is the ideal length. To mark a prove you can use radioactivity, DIG system (using antibodies that recognize some certain molecules that were linked that the synthesis step to the probe, linked to an enzyme that emits light or changes colour with substrate).
Probe labelling We can label the probe with radioactive phosphorous that will enable us to locate the probe with an X-ray film. Nonetheless, radioactivity is rapidly lost and requires specific maintenance protocols that can be a pain in the ass. So, other mechanisms have been developed. Among them the labelling with enzymes whose substrate can be easily spottable (colour or light) or linked to a protein to whom we will direct an antibody.
An example of the last method is the use of Digoxigenin (DIG). One of the nucleotides is linked to that molecule. So by PCR we are able to synthesize probes that contain this molecule.
Afterwards, we can use an antibody (marked in any way) to detect the position of the probe in the Nitrocellulose film.
Pre-Hybridization and Hybridization In the pre-hybridization we prepare the membrane for a proper hybridization of the DNA or mRNA (if it is a Northern). The process’ aim is to block any non-specific interactions that may exist, reducing the background response.
It is achieved after a treatment with a blocking solution with substances that will compete against the probe for non-specific interactions. This way, it’ll be much easier to detect specific signals. As blocking agents, we could use high weight proteins, non-specific DNA, etc... all mixed up with detergent.
Concept of Stringency Is a mixture between Temperature and Salts concentration, that will enable hybridization or not.
The higher the temperature and lower salt molarity, the bigger the stringency will be. And the other way around.
The higher the stringency, the more difficult is for a molecule to hybridize with another.
Salt concentration is important to neutralize the opposing charges of Phosphates in opposing strands. So the higher it is, the more hybridization there will be.
Temperature must be high enough to prevent random hydrogen bonds between non-specific sequences, but not so high to prevent the specific sequences to bind.
By increasing the stringency, we make difficult the nonspecific bonds. But we can’t increase it a lot or the specific bonds will be prevented too.
Plant Biotech Tema 6 Héctor Escribano Southern blot analyssis Hybridization The hybridization solution is the same of the Pre-hybridization (with the blocking agents) but WITH the probe. Before the Hybridization, the probe must be denatured at high temperature (95 ºC) as it is in double strand form, after the synthesis.
To limit the bindings, we make a series of consecutive washes with increasing stringency (diminishing the salt concentration).
Interpreting a Southern gel If a transgene is 2kb and has restriction sites at both sides, it should appear in the gel as 2 kb. But many other things can happen. There might be wrong recombination, that make that fragment smaller, by truncation. There also might be wrong recombination resulting in “concatemere” formation. In this case, the restriction sites might be conversed or not. If all of them are conserved, the fragments after restriction will all be of 2kb.
But if not all of them are conserved, there might appear longer sequences that are also detected by the probe.
Only one restriction site, in the cds, will give us the number of integration sites in the genome.
Because the enzyme will also cut the genome. And there will be as many integration sites as different lengths of digested DNA. Only those labelled by the probe.