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Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method
Biochimica Et Biophysica Acta
|December 13, 1976
Summary
Endonuclease S1 cleavage of hybrid plasmids precisely determines insert length. This method allows for the recovery of globin DNA inserts from poly(dA)-poly(dT) flanked plasmids.
Area of Science:
- Molecular Biology
- Recombinant DNA Technology
Background:
- Hybrid plasmids are crucial for gene cloning and manipulation.
- The poly(dA)-poly(dT) tail method facilitates the insertion of DNA fragments into plasmids.
Purpose of the Study:
- To develop a method for accurately assessing the length of DNA inserts in hybrid plasmids.
- To enable the preparative recovery of specific DNA inserts from these constructs.
Main Methods:
- Cleavage of the hybrid plasmid pBETAG using endonuclease S1 under partial denaturation conditions.
- Analysis of resulting DNA fragments to identify globin-specific sequences.
Main Results:
- The hybrid plasmid was cleaved into two fragments.
- The smaller fragment (575 base pairs) contained the rabbit globin DNA insert.
- Excision occurred within the poly(dA)-poly(dT) regions, indicating the method's specificity.
Conclusions:
- Endonuclease S1 cleavage is an effective method for determining the length of DNA inserts in poly(dA)-poly(dT) flanked hybrid plasmids.
- This technique allows for the preparative isolation of cloned DNA inserts.