Genetic Technologies II

Flashcards for reviewing genetic technologies concepts, including gene cloning, vectors, genomic libraries, cDNA libraries, and PCR.

Plasmid Vector Requirements

Must have an origin of replication (ori), a restriction site for the enzyme used, a selection method for the vector in the cell, and a way to distinguish the vector alone from the vector with the insert.

Polylinker

A collection of unique restriction sites located in the unconserved region of the promoter site for a reporter gene. Insert of DNA destroys promoter; reporter gene is turned off.

Transformation

The process by which bacteria like E. coli are made transiently permeable to DNA in their surroundings, allowing the DNA to enter the cell.

Transduction

The process where DNA is carried into a cell by a phage.

Cell Clones

Cells that arise from an original host cell, each containing multiple copies of a recombinant vector.

Why Clone?

For amplification to produce large amounts of DNA for a typical gene of interest or to produce large amounts of protein from that gene.

Genomic Library

A collection of transformed cells, each carrying a different piece of the genome, collectively representing the organism’s entire genome.

cDNA Library

A library made from complementary DNA (cDNA) that is reverse transcribed from processed mRNA, representing only the genes being expressed in a particular cell type.

Genomic Library Limitations for Protein Production

Genomic DNA contains introns, which bacteria do not know how to remove, preventing proper protein production from eukaryotic genes.

cDNA Library Construction

Involves harvesting tissue, isolating mRNA, using reverse transcriptase to create cDNA, inserting the cDNA into a vector, and transforming host cells.

Genomic Library Representation

Represents the entire genome of an organism.

cDNA Library Representation

Represents only the proteins that were being made in the cells you harvested at the time you harvested them/only represents expressed genes.

PCR Advantages

Speed and relative ease-of-use; it is an all in vitro process.

PCR Limitations

Flanking sequences around the target DNA must be known, and contaminating DNA can be amplified.

What is a DNA library?

A collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study.

How are cell clones Created?

Cell clones arise from an original host cell, each containing multiple copies of a recombinant vector, which is essential for amplifying specific DNA sequences or producing large amounts of protein.

What is the purpose of cloning?

Cloning is performed for amplification, either to produce large amounts of a specific DNA sequence of interest or to generate large quantities of protein from that gene.

What is the representation in cDNA libraries?

In cDNA libraries, the representation includes only the proteins that were being actively produced in the cells at the time of harvest, focusing solely on expressed genes.

What are the key requirements for a plasmid vector?

Essential elements include an origin of replication (ori),