Paula Ripamonte, Giovana Krempel Merighe, Yeda Fumie Watanabe, Alexandre Rodrigues Caetano, Flávio Vieira Meirelles
Published December 7, 2005
Genet. Mol. Res. 4 (4): 726-733 (2005)

About the Authors
Paula Ripamonte, Giovana Krempel Merighe, Yeda Fumie Watanabe, Alexandre Rodrigues Caetano, Flávio Vieira Meirelles

Corresponding author
F.V. Meirelles
Email: meirellf@usp.br

ABSTRACT

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Key words: Differential display PCR, Fluorescence, Universal primer, Bovine embryo.