RESUMO

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

Assuntos

Clonagem Molecular/métodos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genoma Humano/genética , Fator Inibidor de Leucemia/genética , Mucosa Bucal/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Humanos , Fator Inibidor de Leucemia/química , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa