Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes.

AUTOR(ES)

Rohan, R M

RESUMO

When microinjected foreign genes integrate into the genomes of mice, multiple copies are frequently found clustered together at one location. How they concatamerize--by the integration of large linearized concatamers that are formed by simple end-to-end linkage, by circularization of individual DNA fragments and recombination, or by some other means--is not understood. In the transgenic animals studied thus far by ourselves and others, integration frequency and transgene copy number do not seem to be significantly influenced by the complementarity of the ends of the DNA fragments that have been microinjected. We have utilized PCR amplification and DNA sequence analysis to study selected transgene junctions at the nucleotide level. In two transgenic mice carrying the synthetic RSVcat gene (injected with noncomplementary overhangs on the fragment ends), ends were 'nibbled' from 1 to 62 bases before being joined to an adjacent gene copy. Repeated dinucleotides, providing the most minimal of homologies, are present in half of the characterized junctions. Determination of the relative copy number of the junctions in each mouse supports the idea that transgene complexes can undergo additional rearrangements after the initial formation event.

Documentos Relacionados

• Rapid generation of specific single-stranded template DNA from PCR-amplified material.
• A simple and rapid electrophoresis method to detect sequence variation in PCR-amplified DNA fragments.
• Branch migration inhibition in PCR-amplified DNA: homogeneous mutation detection
• Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.
• Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.