Related Experiment Videos
Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing
Abstract:
We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.
Insights
New plasmid vectors enable high-yield single-stranded DNA production for rapid DNA sequencing. These M13 phage-derived plasmids facilitate efficient genetic analysis and molecular biology applications.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- ColE1-derived plasmids and M13 phage are essential tools in molecular biology.
- Efficient production of single-stranded DNA (ssDNA) is crucial for DNA sequencing and genetic manipulation.
- Existing methods for ssDNA production can be inefficient or complex.
Purpose of the Study:
- To construct novel chimeric plasmid vectors for high-yield ssDNA production.
- To enable rapid DNA sequencing using the Sanger method with M13 phage infection.
- To investigate the functional domains of the M13 phage intergenic region.
Main Methods:
- Construction of chimeric plasmid vectors by inserting M13 phage origin and intergenic regions into pBR322.
- Replication of plasmids in the absence of M13 phage (ColE1-like replication).
- M13 phage infection to induce phage-directed plasmid replication and ssDNA production.
- Construction of a deletion derivative (pZ146) to study M13 helper interference.
Main Results:
- Chimeric plasmids (pZ145, pZ150, pZ152) replicate and confer ampicillin and tetracycline resistance.
- M13 phage infection leads to high yields of ssDNA in M13-like particles.
- ssDNA produced is suitable for dideoxy sequencing.
- Deletion derivative pZ146 is encapsidated but does not interfere with M13 helper growth, suggesting distinct functional domains in the intergenic region.
Conclusions:
- The constructed chimeric plasmids are effective tools for producing ssDNA for sequencing.
- The intergenic region of M13 phage contains separate domains for morphogenesis and replication interference.
- These plasmids offer a versatile system for genetic studies and ssDNA manipulation.