[Rapid selection of the gradually shortened DNA clones by using PCR amplification]

To establish a simple technique with which the gradually shortened DNA clones can be selected rapidly and accurately so that the sequencing and identification of the full length cDNA or longer DNA fragments can be performed as quick as possible.

Primers were designed according to the sequence flanking the multiple cloning site of pGEM vector and were used to amplify the inserted fragments in a series of deletion sub-clones originated from a clone. with longer DNA insert fragment. These sub-clones which contain the gradually shortened fragments can be selected directly. This method was compared with the routine method in which the restriction endonuclease was used to digest DNA samples.

A series of deletion sub-clones were identified accurately with this new method and the cycle sequencing could be performed on these PCR products as well. This method was characteristic of more accurate, more simple and less time-consuming, compared with the routine method.

The method presented is very effective for rapid selection of the gradually shortened inserted fragments constructed in pGEM vector. It can greatly save time and materials, and the strategy in the report could also fit for selecting the clones containing gradually shortened fragments in other plasmid vectors.