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Machanical Sharing of DNA

Machanical Sharing of DNA :


Lii addition to digesting DNA with restriction endonucleases to produce discrete fragments, there are a variety of treatments which result in non-specific breakage. Non-specific endonucleases and chemical degradation can be used but the only method that has been much applied to gene manipulation involves mechanical shearing. The long, thin threads which constitute duplex DNA molecules are sufficiently rigid to be very easily broken by shear forces in solution. Intense sonication with ultrasound can reduce the length to about 300 nucleotide pairs. More controlled shearing can be achieved by high-speed Stirring in a blender. Typically, high mol. wt. DNA is sheared to a popuLation of molecules with a mean size of about 8 Kb pairs by stirring at 1500 rev/mm for 30 miii (Wcnsink et a!. 1974). Breakage occurs essentially at random
with respect to DNA sequence producing termini consisting of short single-stranded regions which may have to be taken into account in subsequent joining procedures.

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