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In this article we will discuss about the restriction digestion of DNA and DNA samples.
Restriction Digestion of DNA:
Restriction endonucleases cut DNA molecules into smaller pieces. They cut DNA at the specific recognition sequence, so that it is no longer susceptible to cleavage by the endonucleases. These enzymes are present in vivo in bacteria and are involved in the recognition and destruction of foreign DNA like that of the invading phage. They recognise specific sequences called palindromic sequence in the DNA molecule (whenever that sequence occurs in DNA) and cleave symmetrically both strands.
There are three major types of restriction enzymes; which are I, II and III. Type I & III contain the restriction and modification activities in the same multi-subunit enzyme complex. They require ATP for cleavage and cleave the DNA at substantial distance from the recognition sequence. In contrast, type II endonucleases are not physically associated with the corresponding modification methylases. It does not require ATP for cleavage and generally cleave within or very near
the recognition sequence. Because of this, type II endonucleases are preferred in cleaving specific sites to generate defined fragments. There are more than 600 different commercially available Type II enzymes isolated by molecular biologists. They cut only double stranded DNA and each of the specific sequence is a palindrome. These properties make these enzymes extremely useful in RFLP and DNA finger printing.
Digestion of DNA Samples:
A typical restriction enzyme digestion reaction includes the incubation of a restriction enzyme with DNA in presence of an appropriate buffer and ionic concentrate at a specific temperature and duration.
Requirements:
• DNA samples.
• TE Buffer (pH 8.0)
SDS 10%
Ethanol 95%
• Restriction enzyme.
• Phenol: Chloroform(l:2).
• Chilled ethanol.
• DNA sample and enzyme.
• lOx buffer.
• Centrifuge and tubes.
• Agarose gel.
• Pipettes.
Procedure:
1. To a clean centrifuge tube kept on ice, pipette out:
5 μl of appropriate 10x buffer.
5-10 μg of DNA sample. (Maniatis recommends 1 μg/20 μl)
Water to final volume of 50 μl.
2. Add 1-2 (typically 3 to 20 units) of desired enzyme and mix gently by tapping. The volume of the restriction enzyme added should not exceed 10% of the total volume. If more enzyme is needed, the digestion should be done in a larger volume.
3. Incubate the reaction mixture at the recommended temperature (for most of the enzymes it is 37°C) over night.
4. Run an aliquot of undigested DNA (without enzyme) and digested DNA on a mini agarose gel to ensure complete digestion.
5. If DNA is fully cleaved, the sample can be purified with phenol: chloroform and precipitate the cleaved DNA.
6. Add l/l0th volume of 3(M) Sodium acetate to the reaction mixture and twice the volume of chilled ethanol and keep at —20°C for two hours.
7. Centrifuge at 10,000 rpm for 10 minutes at 4°C.
8. Pour out supernatant and dry pellet.
9. Dissolve pellet in small amount of T.E. buffer and store at 4°C till use.