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Chapter 9
1.) PCR is used to amplify the β-globin gene. The single nucleotide mutation in the gene
that causes sickle cell anemia also abolishes a Cvn1 restriction site. Therefore after the
gene portion isolated and amplified, it is digested with Cvn1 and the fragment are
separated by electrophoresis and visualized with ethidium bromide. If a fragment is
missing on the gel, then the person has sickle cell anemia.
2.) PCR/OLA combines PCR and oligonucleotide ligation assay to detect a single
nucleotide difference. Two short probes are used that are complimentary to the areas
adjacent to the mutation. One probe (X) has its last base at the 3’ end that is
complimentary to the normal nucleotide at the position. The other probe (Y) begins its 5’
end with the nucleotide complimentary to the base adjacent to the site or mutation.
Ligase is next added to the mixture. If the sequence is normal, the two probes will ligate
together. If there is a point mutation, there is no ligation. The ligation is detected using
the labeling of probe X with biotin at its 5’ and probe Y labeled with digoxigenin at its 3’
end. The ligated product has both molecules on its ligated strand. Thus the strand binds
to streptavidin, and the digoxigenin binds to an antibody-alkaline phosphotase that
converts colorless substrate to colored product. The unligated strand only has the biotin
molecule on it and thus does not bind the antibody enzyme complex and there is no color
change.
3.) The ELISA is an enzyme linked immunosorbent assay. In the procedure, the sample
is bound to a support and a primary antibody to the target molecule is added. The
antibody binds to its antigen if present. The support is washed to remove any unbound
antibody. A second antibody is added that is conjugated to an enzyme and it binds to the
primary antibody. The support is washed and a colorless substrate is added. The enzyme
on the secondary antibody catalyzes the formation of a colored product from the colorless
substrate. The color change indicates the presence of the target molecule.
4.) Radioactive dyes are short-lived, potentially dangerous, and require special
laboratory procedures for safe handling. The biotin-streptavidin system is an alternative
system. The biotin is bond to the probe and streptavidin binds to the biotin. Streptavidin
has four biotin binding site per molecule so the other sites are bound with biotin-alkaline
phosphotase compounds. The enzyme either catalyzes a color change or light emitting
reaction. Another method is to use primers labeled at there 5’ ends with a fluorescent dye
that emits light after absorbing light. Finally, molecular beacon probes can be used.
5.) Since the virus is in low levels, I would use a PCR based detection system. I would
determine the sequence of the DNA flanking an identifiable region of specific length in
the virus genome that is specific to that virus and not found in cattle or other variants of
the strain. Using the sequence I would synthesis a probe specific to the regions flanking
the area of interest. Then a tissue sample of the cow could be prepared for PCR and the
method run using the specific primers. The primers would be tagged with a fluorescent
dye fro detection in a capillary electrophoresis genetic analyzer. If a peak came out at the
time corresponding to the fragment length then the virus would be present in the animal.
6.) Specificity means the assay must yield a positive response only for the organism or
molecule of interest. Sensitivity means the assay must be able to identify very small
amounts of the target organism or molecule even with interfering substances.
7.) Chagas disease is detected by the amplification of a 188-bp DNA fragment found in
multiple copies in the T. cruzi genome. The presence of the amplified fragment is
detected using polyacrylamide gel electrophoresis. It could be improved by using
capillary electrophoresis and fluorescent dyes for its detection which is faster, easier and
less variable than gels.
8.) The molecular beacon probe is a non radioactive method for detecting the presence of
a bound probe. The DNA sequence that is complimentary to the target DNA is in
between five nucleotides at each end that are complimentary. One end of the probe has a
flourophore and the other end has a quencher. When the Probe is not bound to the target
DNA the complimentary ends base pair and form a stem. The flourophore and the
quencher are together and the there is no fluorescence. When the probe is bound the stem
is broken up and the quencher does not repress the fluorescence and so it emits light.
9.) DNA fingerprinting makes use of the variability in the human genome that can be
used to confidently distinguish one human from another. To amplify small amounts of
DNA, STR’s are used. A portion of repeated DNA is isolated and primers are designed
to flank the region. Using the primers, PCR amplifies the portion of repeated DNA. This
is down for many loci and the number of repeats a person has distinguishes them from the
rest of the population.
10.) RAPD is random amplified polymorphic DNA. Two short primers of arbitrary
sequence are synthesized and added to the DNA for PCR. When the two primers lay
down on complimentary strands and flank a region, amplified of the segment occurs.
When down with enough primers, this can become specific to the DNA analyzed. IT is
used in plant cultivars to determine if the strains are the same and if they are the will have
the sample amplified DNA portions.
11.) A padlock probe is an oligonucleotide that is complimentary to the target sequence
at its 5’ and 3’ ends but not in the middle. Upon hybridization, the ends pair with the
DNA and are close together and the middle portion loops out. If sequences are exactly
complimentary then the ends of the probe can be joined by ligase, if there is a point
mutation then the strands cannot be joined. The joined probe target can then be detected
with reporter molecules.
12.) Monoclonal antibodies are antibodies that are specific for only one antigenic site on
the molecule. Polyclonal antibodies are a mixture of antibodies for all the different
antigenic sites on the molecule.
13.) The HAT medium contains hypoxanthine, aminopterin, and thymidine and only
hybrid cells can grow on it. Spleen cells or spleen cells fused to themselves cannot grow
in the media. Myeloma cells are HGPRT- and cannot use the hypoxanthine for purine
synthesis and its second pathway is blocked by the aminopterin. The fused cells contain
function HGPRT and contribute it to the fused cell. Thus the fused cell can use the
hypoxanthine to grow in culture.
14.) Molecular beacon probes can be used to detect several genes in the same sample by
using different colored flourophores for each different probe for each gene. They can be
used to characterize a point mutation in sickle cell anemia because if there is a mutant
base pair the probe will not bind correctly and the fluorescence will be quenched.
15.) It is difficult to screen for cystic fibrosis because there are over 500 known
mutations in the CFTR gene that lead to the disease which require that many screening
methods for each mutation.