The screening and identification of recombinant clones varies from one cloning vector to another and from one corresponding host system to another. The screening and identification of recombinant clones varies from one cloning vector to another and from one corresponding host system to another. Theoretically, the screening of recombinant clones is to exclude clones formed by vectors that have cyclized themselves, vectors that have not been completely enzymatically cleaved, and vectors with non-target DNA fragment insertions. There are two types of screening methods commonly used. One type is the screening method for genetic phenotypic alterations, represented bsy the β-galactosidase system screening method. The other type is the screening method that analyzes the structural characteristics of recombinants, including rapid lysis of colonies to identify plasmid size, restriction enzyme mapping identification, Southern blot hybridization, PCR, colony (or phage spot) in situ hybridization and other methods.
1. β-galactosidase system screening method (blue-white spot screening method)
The vectors using this method include M13 phage, pUC plasmid series, pGEM plasmid series, etc. The common feature of these vectors is that the vector carries a segment of the bacterial gene lacZ.
2. Rapid lysis of colonies to identify plasmid size
The colonies are picked from the plate, incubated overnight and lysed, and then directly subjected to gel electrophoresis, and compared with the vector DNA to determine the presence of the insert fragment based on the decrease in mobility. This method is suitable for the initial screening of recombinants with large insertion fragments.
3. Restriction enzyme profile identification
For the initial screening of colonies with recombinants, purify recombinant plasmid or recombinant phage DNA, cut the insert fragment released from recombinants with corresponding restriction endonucleases (one or two), for recombinants with possible bidirectional insertion, also use appropriate restriction endonuclease digestion to identify the insertion direction, and then use gel electrophoresis to detect the size of insert fragment and vector.
4. Southern blot hybridization
To determine the correctness of the DNA insertion, after digestion of the recombinant by restriction endonuclease and separation by gel electrophoresis, the DNA is transferred to the nitrocellulose membrane by Southern blotting, and then the corresponding exogenous DNA fragments, either radioisotopically or non-radiolabelled, are used as probes for molecular hybridization to identify whether the insertion in the recombinant is the desired target gene fragment.
5. PCR method
Analysis of recombinants by PCR not only allows for rapid amplification of the insert fragment, but also allows for direct DNA sequence analysis.
6. Colony (or phage spot) in situ hybridization
Colony or phage spot in situ hybridization technique involves transferring transformed bacterial DNA to nitrocellulose membranes, molecular hybridization with specific DNA or RNA probes that are radioisotopically or non-radiolabeled, and then selecting positive clones. This method allows for large-scale manipulation and is the preferred method for screening gene libraries.
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