Molecular Biology Question

[letaps]

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Hey,

What is the purpose of DNA digestion using restriction enzymes and what are the applications of DNA digestions (meaning, why do we perform digestions)? Can you give me some examples?

Thanks

 

[tncekm]

You're fragmenting the DNA at known sequences, so you sort of know where you're cutting it.

An example would be, say you've extracted some mRNA, reverse transcribed, and made cDNA and want to see if it contains a gene. IF you know that gene has 1,500 base pairs between two different restriction locations (assuming they only occur once in the plasmid/chromosome, whcih is unlikely but humor me for this example) then you can digest the gene, perform agarose gel electrophoresis with the sample against a known ladder, and see if you've got a 1,500 base pair fragment. If so, then you know you might have that gene you're looking for and may feel like having it sequenced to confirm the identity.

 

[NontradICUdoc]

You're fragmenting the DNA at known sequences, so you sort of know where you're cutting it.

An example would be, say you've extracted some mRNA, reverse transcribed, and made cDNA and want to see if it contains a gene. IF you know that gene has 1,500 base pairs between two different restriction locations (assuming they only occur once in the plasmid/chromosome, whcih is unlikely but humor me for this example) then you can digest the gene, perform agarose gel electrophoresis with the sample against a known ladder, and see if you've got a 1,500 base pair fragment. If so, then you know you might have that gene you're looking for and may feel like having it sequenced to confirm the identity.

Well said. There also needs a little more clarification. The restriction enzymes that the above poster mentions also have to be unique. For example, if you have a segment that has and EcoRI site and then 1500 base pairs later it has a HINDIII site, then you can use it for that. In addition, you can use restriction digestion to insert specific genes into a plasmid. If you have an enzyme that creates sticky ends then you can cut the plasmid and the DNA fragment with the same enzyme and then ligate the enzyme into the plasmid.

Restriction digestion is also used for Paternity Testing to see if the child has the same RFLPs as the mother and the father.

 

[tncekm]

Good info.

IMHO this topic is best explained w/ pics though. Sometimes its difficult for people to visualize what's going on.

 

[letaps]

Can you guys post any links that show good pics that explain this topic in an easy to understand manner?

Thanks