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Biology Tools

In: Science

Submitted By hassanpuri
Words 1029
Pages 5
Biotechnology tools and Techniques
1) Restriction enzymes
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.
In bacteria the restriction enzymes naturally provide immunity to the bacteria against bacteriphage by cutting viral DNA is that introduced into the cell (Fig. 2 Page 280).
After being cut, the viral DNA becomes inactive and thus the phage cannot attack the bacterial cell.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides known as recognition sites.

Most recognition sites are 4 to 8 base pairs long resulting in relatively low frequency of cuts compared to 2 base pair recognition sites.

Example finding a 6 base pair recognition site for EcoRI is 46 i.e. once in every 4096 nucleotides. Whereas the probability of finding 2 specific bases next to each other is 42, once in every 16 nucleotides.

More frequent cuts due to shorter recognition site results in a better chance to isolate a gene of interest.

Recognition sites are characterized by a complementary palindromic sequence

For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5'GGCC3' 3'CCGG5'
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined.

(Copy Table 1 on page 279 for more examples of enzymes and their recognition sites)

The cuts produced differ based on where the phosphodiester bonds are broken in the recognition sites.
HaeIII

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