Double Stranded DNA 'Forward' vs 'Reverse' Orientation PDF
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Uploaded by SaneHilbert
University of St. Thomas (TX)
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Summary
This document explains the concepts of forward and reverse orientations in double-stranded DNA. It provides examples of DNA sequences with EcoRI restriction sites and details how mRNA synthesis and protein production differ in the two orientations. The document is focused on molecular biology concepts for higher education levels.
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Double Stranded DNA ‘Forward’ vs ‘Reverse’ Orientation Below is an example of the dsDNA that corresponds to the protein coding region (ie, starting with a start codon and ending with a stop codon – all other elements have been removed to facilitate visualization). The coding region is surround...
Double Stranded DNA ‘Forward’ vs ‘Reverse’ Orientation Below is an example of the dsDNA that corresponds to the protein coding region (ie, starting with a start codon and ending with a stop codon – all other elements have been removed to facilitate visualization). The coding region is surrounded by 6 ‘N’ nucleotides and also flanked by an EcoRI restriction site. 5’ GAATTC NNN NNN ATG GGA CAG GAA CAG GAT ACA CCA TGA NNN NNN GAATTC 3’ 5’ GAATTC NNN NNN TCA TGG TGT ATC CTG TTC CTG TCC CAT NNN NNN GAATTC 3’ When we treat this fragment with restriction enzyme EcoRI, we obtain a 5’ overhang: 5’ AATTC NNN NNN ATG GGA CAG GAA CAG GAT ACA CCA TGA NNN NNN G 3’ 3’ 5’ AATTC NNN NNN TCA TGG TGT ATC CTG TTC CTG TCC CAT NNN NNN G If the cloning vector is cut I the lacZ gene with the same restriction enzyme, we would expect the circular DNA to open as show below: Assuming the promoter is to the left of the restriction site on the vector, the top strand would be the coding strand, and the bottom strand would be the template strand. Below are the two ways that the sticky-ends of our DNA fragment of interest can lineup with with those of the vector. In each case, the mRNA and protein are show. Forward orientation: protein is synthesized AATTC NNN NNN ATG GGA CAG GAA CAG GAT ACA CCA TGA NNN NNN G AATTC NNN NNN TCA TGG TGT ATC CTG TTC CTG TCC CAT NNN NNN G mRNA: 5’ GAAUUC NNN NNN AUG GGA CAG GAA CAG GAU ACA CCA UGA NNN NNN GAAUUC 3’ The resulting protein would be: MGQEQDTP* Reverse orientation: NO protein can be synthesized AATTC NNN NNN TCA TGG TGT ATC CTG TTC CTG TCC CAT NNN NNN G AATTC NNN NNN ATG GGA CAG GAA CAG GAT ACA CCA TGA NNN NNN G MRNA: 5’ AATTC NNN NNN UCA UGG UGU AUC CUG UUC CUG UCC CAU NNN NNN G
