抽象-哥伦比亚大学（ abstract- columbia university）.docVIP

Biotechnology Homework 2 Fall 2011 Hand in on 9/28 Please read questions carefully. You always need to provide explanations in your answers. 1. This question concerns some basic plasmid cloning procedures and design choices. The overall task is realistic. You have two pure (cloned) DNAs as starting materials. One is the cDNA for a particular mouse gene in an ampicillin resistant vector. A full-length cDNA is a complete copy of an mRNA and therefore has sequences corresponding to 5’ untranslated sequence (5’ UTR), coding region (starting with the ATG triplet [AUG in mRNA] and ending with a stop codon) and 3’ UTR. The second DNA (also an ampicillin resistant plasmid) is a mouse expression vector. It has a DNA segment that acts as a strong promoter in the mouse tissue culture cells, followed by a sequence that directs the initiation of translation and encodes a short epitope tag sequence of amino acids. Beyond the epitope tag segment are sequences that will form 3’ UTR and direct polyadenylation of the transcribed RNA (polyA signal sequences). In mouse cells the promoter will direct transcription to start before the epitope tag sequence and the latter segment will direct translation of the resulting mRNA initiating at the ATG as diagrammed (next page). The sequence of the first few amino acids and corresponding codons is shown. This short stretch of amino acids (this particular epitope tag is known as a Flag tag) is used because it can be recognized by a commercially available antibody. If a cDNA segment is inserted appropriately between the epitope tag sequence and the polyA signal sequence and the resulting DNA is introduced into mouse cells an mRNA should be produced that is translated into a fusion protein containing the epitope tag at its N-terminus followed by the amino acid sequence encoded by the cDNA. This requires that the mouse cDNA sequence is translated in the correct reading frame and contains the entire coding region. Neither the 5’UT