Hall Book Ch 17 (Molecular Techniques in Radiobiology) Flashcards
Restriction endonucleases are enzymes that cleave DNA after recognizing a specific sequence. Those most useful for constructing recombinant molecules leave a “sticky” end, that is, a single-strand overhang of two to four nucleotides that can pair with a complementary strand.
A vector is an autonomously replicating DNA molecule into which foreign DNA fragments are inserted and then propagated in a host cell. Vectors include plasmids, bacteriophage, BACs, and viruses.
A library is a collection of cells, usually bacteria or yeast, containing recombinant vectors, carrying DNA inserts from a different species. The
inserts may be constructed by using restriction enzyme–digested genomic
DNA or cDNA.
A host is used to grow (i.e., to multiply) or to express a DNA fragment
containing a GOI.
In agarose gel electrophoresis, DNA or RNA molecules can be separated
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according to size by causing them to move through a matrix composed of
purified agar under the influence of an electrical field.
PCR is a procedure that enzymatically amplifies the number of copies of a
DNA sequence, up to several thousand base pairs, through repeated
replication by DNA polymerase. (A DNA polymerase is an enzyme that
catalyzes the addition of nucleotides to a growing DNA molecule.) PCR has
been adapted to analyze RNA expression through the use of RT-PCR. Use of
a specialized thermocycler enables qRT-PCR, a very sensitive PCR technique
that enables high-throughput nucleic acid analysis and quantitation.
CGH is a more modern refinement that starts by identifying regions of
chromosomal duplication or deletion. The genes in the abnormal region can
then be analyzed by expression or functional studies to identify candidate
genes in the region.
RNA transcription can be measured by Northern blotting, RT-PCR methods,
hybridization to microarrays, or RNA-Seq.
The microarray is a useful platform for studying gene expression changes
(RNA), gene aneuploidy (CGH), and protein–DNA interactions (ChIPChips). The primary advantage of the microarray is the sheer number of
sequences that can be analyzed at one time. The major disadvantage is a
heavy reliance on knowing genome sequence.
The completion of whole genome-sequencing projects for humans, mice, fruit
flies, nematodes, and yeast has made gene mapping largely obsolete.
Virtually any genomic sequence can now be accessed from international
databases.
Once a gene is cloned, it can be sequenced by one of several methods, the most
common of which is the chain termination method. Use of fluorescently
labeled dyes on the dideoxynucleotides enables automated laser scanners to
acquire sequence from a single lane.
The amino acid sequences within a protein can be determined from the
corresponding DNA sequence using the known genetic triplet code.
A newly acquired sequence can be compared with the international database to
seek homologies with known genes or proteins.
Structural variations in DNA caused by point mutations, deletions, or
insertions can result in restriction fragments of different lengths, which can
often be detected by Southern blotting. These are known as restriction
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fragment length polymorphisms, which can be used as genetic markers to
map genes to specific chromosomal locations and identify aberrant genes
causing disease.
A mutation involving only a single base-pair difference between two short
single-stranded DNA molecules can be detected by the technique of singlestranded conformation polymorphism.
A useful approach to studying a GOI is to introduce it into a cell or organism
(typically a mouse), overexpress it, and observe the biologic effect.
Another useful approach for studying the function of an identified gene is to
“knockout” expression of a gene. This can be done by using either
homologous recombination or RNAi.
CRISPR-Cas9 is a powerful new tool in genome editing, which requires three
components—a Cas9 nuclease with its CRISPR RNA and trRNA.
Commercial versions have combined all of these components into a synthetic
guide RNA.
RNA-Seq exploits deep sequencing to obtain information on both transcript
structure as well as transcript number.
Genetic reporters are useful tools for studying biologic phenomena like
transcriptional regulation, protein dynamics, and intracellular location.
Reporters have easily assayable activities (light emission, fluorescence, or
colorimetric substrates), providing flexibility to suit a given experimental
system.
The ChIP assay can be used to study promoter function in intact cells, either to
identify transcription factor interactions with promoters or to dissect
mechanisms of regulation. DNA purified by ChIP can be analyzed
individually by PCR or by hybridization to promoter microarrays.
The field of proteomics seeks to define the quantities and interactions of the
vast number of proteins in a given cell at a given instant in time. Proteomics
uses established technology like 2-D electrophoresis, Western blotting,
immunoprecipitation, and mass spectroscopy but uses more advanced highthroughput technology to expedite the process.
Protein–protein interactions can be identified by coimmunoprecipitation or
two-hybrid analysis. Fluorescent protein fusion proteins are useful tools for
further study of intracellular interactions and dynamics.
