Biochemistry DNA hybridization question

[dex.z]

This is my first post. so apologies for breaking any posting rules.

 

My question regards a pre hybridization procedure in which the denatured DNA is transferred onto a nylon membrane. To do this the nylon membrane is baked which allows the DNA to covalently bind to the membrane, through a dehydration reaction. Or so I was told by my university tutor. However, I'm not sure of the chemistry behind this reaction.

 

If anyone could explain or reference a source that can explains the chemistry concisely I would appreciate it. Thanks.

[Popcorn Sutton]

Baking it might not be the most efficient method, although it may achieve some degree of success, but if that were the case, that would mean that we are becoming what we eat. There are several ways in which you can make a hybrid. All logical, but people may not want me to share that information.

Edited April 9, 2013 by Popcorn Sutton

[CharonY]

Popcorn, your answer does not even begin to make sense. For the most part I would think that typical ways to immobilize DNA on nylon is blotting under alkaline conditions (the positive charges of the nylon are sufficient to retain the DNA) or cross-link with UV. Vacuum Baking is a bit more old-fashioned way and I am not even sure that the precise nature of the interaction between nylon and the nucleic acid has been well understood (or investigated). It is generally not assumed to be a covalent bond, but something involving salt bridges and hydrophobic interactions (for nitrocellulose that is; Schleicher and Schuell 1992). Which basically means that these are rough guesses.

Edited April 9, 2013 by CharonY

[Popcorn Sutton]

Are you guys actually talking about the fabric nylon? That would not make much sense to me, where does nylon become useful with dna? I was talking about efficient methods of merging dna although im not aware of how to target specific genomes. I know the materials one would need to merge dna from two species and I know the process to get the right effect. But like I said, I'm interested in learning how to target and isolate genes.

 

I thought the OP was about merging dna.

 

I guess a method you could use to bond dna with nylon is by forming a culture, spreading it across the nylon, and using something to isolate the dna. Alcohol is a possibility, but I don't know if that would damage the dna. I wouldn't want to burn the dna, it seems like it would fragment the sequence. Burning the nylon might be logical to increase the surface area so it catches more dna.

 

Electricity is useful. I hear that using 1.5v is efficient for making the membrane permeable (a D battery)

Edited April 9, 2013 by Popcorn Sutton

[CharonY]

The OP was talking about a fairly standard method in molecular biology, termed blotting (specifically the fixation step after that). Blotting, in turn is a standard procedure used during Southern hybridization. I appreciate the fact that you would like to join discussions, but throwing wild assumptions around without actually understanding the topic at hand can be amusing for some readers, but especially for newcomers it may be a tad confusing. It certainly is not helpful...

[dex.z]

thanks for the responses. But im still not entirely sure how vacuum baking causes the dna to be fixed to the nylon membrane. Im aware of other methods (such as UV crosslinking) but for my particular case I have to explain how it happens in terms of heating the membrane to 80C for 30 mins.

From this site:
http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/northern-analysis/tech-notes/membrane-transfer-and-crosslinking-for-rna.html

 

They give the following explanation:

Baking

Baking works by heating the membrane to drive out all water solubilizing the RNA. A large component of RNA is its hydrophobic nucleotide bases, which make hydrophobic contacts with aromatic groups on the membrane. This interaction is affected by heating in an oven at 80°C for 15 min. The only danger in baking is that the membrane can be damaged if the heat is not regulated to prevent temperatures from rising much higher than 100°C.

Is it safe to assume single stranded DNA undergoes similar process? Since the only difference between RNA and single stranded DNA is in the ribose which doesn't seem to have an importance in its fixation to the membrane..

[BabcockHall]

The explanation may have some truth in it, but I have to wonder whether or not the whole story is exactly what they imply. The interactions among the bases in ds DNA is not classically hydrophobic in nature, if I recall what I read in Voet and Voet correctly. Of course, the interactions between aromatic groups on the membrane with the bases of DNA or RNA might still be hydrophobic, but this should ideally be demonstrated.

[CharonY]

As I mentioned, I do not think that anyone has investigated the precise physics behind it in detail. The best I found was more or less a list of potential interactions with all the usual candidates (with hydrophobic interactions being near the top of the list).

There are some models around, mostly from the manufacturers as e.g.: http://www.pall.com/main/oem-materials-and-devices/literature-library-details.page?id=27294.