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Unit 11 Gene Mutations
Helpful Hints
Molecular diagnostics is being revolutionized by the completion of the human genome
project and by the development of highly advanced technologies for DNA testing.
Mutations can be caused in a variety of ways, from a single substitution in a base pair to
large changes in DNA which will have a significant effect on the protein sequence. Base
substitutions (aka point mutations) occur when 1 base on a DNA strand is replaced with a
different one. This can affect the resulting protein in various ways. Great distortions can
arise because during transcription and translation, the DNA that is being read is wrong,
and thus do not correctly produce a needed protein. An incorrect sequence of amino acids
may alter the shape of the protein, the most important characteristic that defines a
protein’s function. In the case of sickle cell anemia, the hemoglobin molecule is
crystalline and makes the red blood cell crescent-shaped. Even a slight change in the
sequence may be enough to completely alter the information.
Molecular methods are designed to detect these mutations. The most popular methods
incorporate fluorescent dyes to visualize and/or detect the target. Simply put, probes
have the dye incorporated into them. When the labeled probe binds with their target
fluorescence occurs. We can detect a single target with one probe, a large target with
probes that flank the target region, or use 1 or 2 probes directed against the target on the
complementary strands, since the mutation may only be detected on one strand of the two
DNA strands.
Testing requires 4 constituents: a target, probes, label(s) and a detection system.
Although the focus in this chapter is the detection of genetic mutations the methods
presented can also be utilized in testing protocols used to detect a specific sequence of
genetic material such as testing for HIV, blood grouping antigens, tumor cells, bacteria
and so on. Primers can be built for anything with a genetic code.
With this in mind, review chapter 9. Use the vocabulary list to learn the language of
these methods. Do not focus on the in depth details of each method presented, but the
basic principle of the test, clinical use (screening or diagnostic), and whether this
procedure is used routinely in the clinical laboratory. As you read through the chapter
refer to the diagrams often. My goal is that you have a basic understanding of the
CONCEPTS of the testing, not the minutiae, ie, the step by step process of performing
the tests. Complete the chart I have posted in the “Objectives” section as you will be able
to use this as a “cheat sheet” for Exam 3.