Hubbard, Christopher J.
B.S. (Bachelor of Science)
Department of Biological Sciences
Studies have been done on the decay rate of messenger RNA (mRNA), including the decay rate of EGF-R mRNA. Several factors in the 3’ untranslated region (UTR) have been suspected to affect the mRNA decay rate. However, no experiments have been done to determine exactly what portion of the 3’ UTR controls the decay rate. In order to study the effects of segments of 3’ UTR, it is necessary to start with EGF-R and complete 3’ UTR. The focus of this cloning procedure is to produce the EGF-R cDNA with complete 3* UTR. Copy DNA (cDNA) is single stranded DNA that is complementary to an RNA, synthesized by the enzyme reverse transcriptase (in vitro). The cDNA contains only the exons of the gene. The EGF-R cDNA refers to the EGF-R cDNA and approximately 150 base pairs (bp) of the 3’ untranslated region (UTR). The 3’ UTR is the region that extends from the stop codon (termination of protein synthesis) to the start of the poly A tail. In this experiment, the EGF-R cDNA and entire 3’ UTR are first isolated. The 150 bp of 3* UTR on the EGF-R cDNA is removed. The EGF-R cDNA (without the 150 bp of 3* UTR) and the entire 3* UTR are then ligated together, and inserted into a vector (i.e. plasmid). This hybrid plasmid will be inserted into a cell line by a process called transfection. The stable transfection process results in cells that have incorporated the EGF-R cDNA (with complete 3* UTR) into the cell's genome. This allows multiple copies of the gene to be made. The cells are lysed, and the DNA can be isolated for study.
Lunkenheimer, Rod, "Cloning strategy for developing a complete EGF-R cDNA" (1997). Honors Capstones. 297.
17 unnumbered pages
Northern Illinois University
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