Molecular Biology: DNA sequencing
Molecular Biology:
DNA sequencing
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.
APPLICATIONS
There are many applications for sequence data. The following are just a few:
Diagnostics
The DNA sequences of disease-causing organisms can be used to assist in the diagnosis of
veterinary diseases. Sequence data can be particularly useful for the identification of microbial
disease-causing organisms such as viruses, bacteria and protozoa. Frequently many closely related
species are present in the field, only some of which cause disease, and it is often difficult to
distinguish between these species in any other way.
One of the most widely used loci in diagnosis is the ribosomal RNA operon. Portions of the nucleotide
sequence of rRNA genes are highly conserved between all organisms, particularly in regions which
determine the secondary structure of the ribosome. Other regions of rRNA genes, which are not
under pressure to remain unchanged, vary between species. The structure of rRNA genes is
therefore ideal for the development of a PCR test to distinguish between related species. Primers can
be designed in conserved areas; these primers will amplify a portion of the rRNA gene from all related
parasites. Very specific and sensitive probes can then be designed in the variable regions of the
rRNA genes to distinguish between the parasites.
Phylogenetics
Phylogenies can be used to determine evolutionary relationships between organisms. Such
relationships can assist in epidemiological studies and can be used to determine the origin of isolates
in outbreaks of certain diseases.
Phylogenetic analyses that are based on sequence data depend upon initial homology inferences at
the amino acid or nucleotide level. Thus, the accuracy of phylogenies will depend upon this first step.
The most valuable phylogenetic information can be obtained from amino acid sequence alignments. It
makes little sense to align untranslated nucleotide sequences of coding regions, because of codon
degeneracy (codon variability is higher than amino acid variability). The exception is if one is looking
for variation within the nucleotide patterns themselves, as would be the case with tRNA or rRNA
genes, promoter regions or terminators. In these cases the same methods that are used to align
amino acids can be applied.
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Molecular Biology: DNA sequencing
Microsatellites
Microsatellites are stretches of DNA that consist of tandem repeats of a simple sequence of
nucleotides. The repeat units are generally di-, tri- tetra- or pentanucleotides (for example, AAT
repeated 15 times in succession). Microsatellites tend to occur in non-coding DNA. Microsatellites
form through slipped-strand mispairing at replication pauses or single strand annealing following
exonucleolytic degradation at a DNA double-strand break. These processes can result in variable
numbers of repeat units and thus microsatellites tend to be highly polymorphic.
Microsatellites are useful genetic markers precisely because they are polymorphic, and in addition
they are locus-specific. PCR primers can be designed in the regions flanking the repeats and using
the polymerase chain reaction (PCR), the microsatellites can be easily amplified. The number of
repeat units that an individual has at a given locus can be easily resolved using polyacrylamide gels
or in capillaries containing a liquid polymer.
There are numerous applications for microsatellites. They have been used as markers for certain
disease conditions. Some microsatellite alleles are associated, through genetic linkage, with certain
mutations in coding regions of the DNA that can cause a variety of medical disorders, such as
schizophrenia (Bailer et al., 2002). Because of their high specificity, microsatellites are frequently
used as forensic markers to test DNA in court cases, and have been used to identify both human and
animal DNA samples. DNA from the crime scene is compared with DNA from the suspect. Match
identities for microsatellite profiles can be very high – in fact, the probability that the evidence from the
crime scene is not a match with that of the suspect is less than one in many millions in most cases.
Parentage analysis is another application for microsatellites. Each individual inherits one length of
nucleotide repeats from his or her mother and one from his or her father (individuals with a single
band received the same band from both their mother and their father). For captive or endangered
species microsatellites can serve as tools to evaluate inbreeding levels.
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