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Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail
H Tröster1, W Kissel, M F Trendelenburg
1Institute of Experimental Pathology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Molecular & General Genetics : MGG
|June 1, 1989
Summary
Lambda EMBL3 cloning revealed its left arm within the BamHI site, forming palindromic sequences. This structure hinders BamHI enzyme cleavage, impacting recombinant DNA analysis.
Area of Science:
- Molecular Biology
- Recombinant DNA Technology
Background:
- Lambda EMBL3 is a widely used bacteriophage vector for cloning.
- Understanding vector structure is crucial for efficient gene cloning.
Purpose of the Study:
- To analyze the structure of recombinant lambda EMBL3 clones.
- To investigate the behavior of the BamHI cloning site in lambda EMBL3.
Main Methods:
- Restriction enzyme digestion analysis (KpnI, BamHI, SalI, EcoRI, HindIII).
- Analysis of eight representative recombinant lambda EMBL3 clones.
Main Results:
- Lambda EMBL3 contains its own left arm within the BamHI cloning site.
- Recombinant molecules were generated and viable in Escherichia coli NM539.
- A palindromic sequence formed at the ligated left and right arms, hindering BamHI cleavage.
- Incomplete BamHI cleavage suggests a cruciform DNA structure.
Conclusions:
- The inherent structure of lambda EMBL3 influences cloning efficiency.
- A cruciform structure at the BamHI site may impede restriction enzyme activity.
- Further investigation into DNA secondary structures in cloning vectors is warranted.