transgenics and gmos- lecture 5 Flashcards
transgenic
an organism whose genome has been altered by the transfer of a gene or genes from another species or breed (sometimes called genetically modified, gmos)
when disturbed, aequrea produces green light, caused by
green fluorescent protein (gfp)
cloning
take piece of dna and park it into a bacterial cell
*take plasmid out of bacterial cell, put little fragments of jellyfish dna into the plasmid, then park the plasmid back into the bacterial cell
*persuade bacterium to express whatever is in the plasmid
*expression assay to detect successful colony, plasmid with gfp gene will glow green
plasmid
little loop of dna that can be semi independent like a second chromosome in a bacterial cell
genomic library
in eukaryotic genes, amino acid coding exons are often interrupted by non coding introns
cdna
complementary dna- converting jellyfish edited mrna into dna
creating a cdna library
need an enzyme to convert dna from rna, reverse transcription
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription.
reverse transcriptase process
1) reverse transcriptase needs a primer, and mrna has a poly a tail so primers made of thymine are used
2) reverse transcriptase leaves a small 3’ overhang, then used as a primer for dna polymerase which uses single stranded dna as a template to produce double stranded dna
3) s1 nuclease is used to cleave the bend in the dna,, leaving double stranded dna with blunt ends
to allow us to park our cnda into a plasmid, we need two enzyme tools
restriction enzyme and a ligase
expression vector
has the material necessary for expression that is not present in the gfp sequence
getting the plasmid into the bacterial cell: electroporation
electroporation causes components of the membrane to become fluid which allows things to squeeze in from the outside
how do we find the bacteria with the cdna we want
pcr cloning
a strategy for cloning a coding sequence by taking advantage of the known sequence of a cdna to simplify it via pcr before inserting this product into a plasmid
hybrid primers
part restriction enyzme recognition sequence and part gfp sequence complement
what determines when and where a gene is expressed
promoter (through interaction with regulatory molecules like activators and repressors) because where the promoter is, that is where the transcription factor binds
genetically modified organisms/transgenic organisms
any organism whose genome has been altered by the insertion of a gene or genes from other species or breeds
refers to the isolation of a DNA sequence from any species (often a gene), and its insertion into a vector for propagation, without alteration of the original DNA sequence
molecular cloning
expression cloning
In this technique, researchers isolate all of the
genes that are being expressed in an organism, and test them one by one for a function of interest. We can explore this technique by walking through its steps for identifying the gene encoding green fluorescent
protein, GFP
Reverse transcription is a process by which researchers
can make DNA copies of RNA molecules using the enzyme:
reverse transcriptase
how does reverse transcriptase start dna synthesis
To start the DNA synthesis, reverse transcriptase requires a primer, which is a short oligonucleotide that specifically binds to the RNA of interest. It is especially easy to reverse transcribe eukaryotic mRNAs,
because they all share a common sequence feature that can be used to facilitate primer design – the poly-A tail. Since every mRNA has a poly-A tail at its 3’ end, an oligo (dT) primer, a primer consisting of only thymine
residues, can be used to synthesize a complementary strand of DNA using the mRNA as the template
how do rna molecules perform a reverse transcriptase reaction
To perform an RT reaction, RNA molecules are first isolated from cells, and mixed with the oligo (dT) primers. Each primer will bind to the poly-A tail of a different mRNA, allowing the reverse transcriptase enzyme to bind the template and synthesize a strand of DNA with sequence complementary to the RNA. This generates a double-stranded RNA-DNA hybrid. In addition, the RT process adds a few additional bases to the 3’ end of the new DNA strand. This short extra piece of DNA then serves as the primer for synthesizing a second strand of DNA using the first as a
template.