Biomedical Engineering Reference
In-Depth Information
of plasmid from which minus strand (and thus “antisense”) RNA is syn-
thesized, or by introducing single stranded cDNA oligonucleotides into
the cell. The theory is that the antisense RNA or cDNA forms duplexes
with the mRNA and consequently inhibits translation. This is thought to
be accomplished by preventing the ribosome from gaining access to the
mRNA or by increasing the rate of degradation of the duplexed mRNA.
In either event, the failure to translate the protein provides an oppor-
tunity for an investigator to test for changes in the cell that result from
the lack of gene expression. Obviously, this method works only if the
endogenous protein in the cell is relatively short-lived!
RNA interference (RNAi)
RNAi is a naturally occurring phenomenon but can also be used as
a method of gene silencing. RNAi has been used in a number of in-
tact organism including the nematode C. elegans, where this method
was first discovered, as well as in fruit flies and plants. RNAi begins
with long, double-stranded RNA molecules (usually larger than 200 nt),
which are enzymatically cleaved by an RNase called “dicer” in the cell
to 22 bp molecules, which are called
s
mall
i
nterfering
RNAs
(siRNA).
The RNA is assembled into complexes that lead to the unwinding of the
siRNA, which hybridizes with mRNA and inhibits its translation, presum-
ably by causing its cleavage and destruction. Molecular details of these
processes remain poorly understood.
RNAi does not work effectively in mammalian cells because the in-
troduction of dsRNA into these cells can lead to an antiviral response
due to the recognition of the RNA by certain receptors involved in the
innate immune response, called Toll-like receptors (TLR). However, the
introduction of the shorter siRNA into some, but not all, mammalian cells
can lead to gene silencing. In these cells this provides a potent means
to study gene function in these cells.
I.
Forensics and DNA technology
Introduction
Most of us are aware of real-life situations where DNA typing has been
used to attempt to solve a murder (who can forget “the OJ case”?).
The significant use of DNA techniques has even made it into our en-
tertainment culture, as exemplified by the popularity of TV programs
like the “CSI: Crime Scene Investigation” series. But what methods are
used for identification of DNA samples to match them to a particular
individual?
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