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18 Cards in this Set

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what are the names of the two techniques used to measure levels of mRNA of specific genes? What is the major difference between the 2?
Northern blots and microarrays; Northern blots are used to measure mRNA levels for a single gene at a time while microarrays are used to measure mRNA levels for thousands of genes at the same time
in addition to expression of mRNA for a given gene in given tissues, what information about the mRNA does the Northern blot provide?
the relative size
what 3 things are required to create radioactive copies of a cloned gene in vitro?
DNA polymerase, primers, and radioactive dNTP's
what must occur following creation of double-stranded radioactive DNA clone for Northern blot to proceed?
DNA must be denatured to form single stranded copies
what is the name for the single-stranded radioactive DNA used for Northern blots?
probe
how is mRNA prepared for a Northern blot?
RNA is isolated from cell or tissue then fractitioned by size using agarose gel electrophoresis
what must occur once mRNA has been run on agarose gel?
RNA is transferred to nylon or nitrocellulose membrane using capillary action
membrane containing RNA is incubated with radioactive DNA probe under conditions that favor what process?
hydrogen bonding
the what is the name for the signal produced by radioactive decay of probe when exposed to X-ray film?
autoradiograph
what does the intensity of the signal on an autoradiograph represent?
how much RNA is present that matches the DNA probe in a given tissue
an array used for microarray analyses is composed of which type of fragments?
cloned DNA fragments
array is hybridized with what in microarray analyses?
mRNA from various tissues
how are cDNA fragments from different tissues differentiated in a microarray analysis?
they are tagged with different fluorescent molecules so mRNA expressed in one tissue is one color and mRNA from another tissue is another color. mRNA expressed in both is a color which combines both tissues.
None
how is microarray technology useful for cancer assessment?
RNA levels of specific predictor genes are determined for all tumors and this gene expression profile will be used to determine how to treat tumors.
how is the issue of lack of bacterial promoter in desired eukaryotic genes overcome in the protein expression in bacteria? Which promoter is most commonly used? How can this promoter be regulated?
genes are inserted downstream from a bacterial promoter; lac promoter; IPTG which is a lactose analog
how is the issue of lack of splicing in bacterial protein synthesis overcome in the production of eukaryotic proteins in bacteria?
iit is necessary to use cDNA and not genomic DNA when making proteins in a bacterium. In this way, the introns have already been removed and the mRNA will not need to be modified following transcription.
how are eukaryotic genes inserted such that they produce a Shine Delgarno sequence for translation?
they inserted into an existing bacterial gene so that the translation reading frame between the mammalian gene and the bacterial gene are fused.
what is another advantage of creating a fusion protein?
provides an affinity tag that can be used for easy purification of the protein.