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Diagnosing Lupus With PCR
Systemic lupus erythematosus (SLE) is characterized by exacerbated inflammatory responses
throughout the body, suggesting a possible aberrant complement reaction. Once the genes
responsible for the components of the complement cascade were discovered and localized,
scientists were able to analyze a possible link to SLE. One means to establish a correlation between
genes and disease is to compare gene variations among normal and afflicted individuals. Family
pedigrees readily permit these kinds of investigations. In a very simplistic example, suppose that a
given gene responsible for a complement component is compared among members of a family,
some of whom are afflicted with an immune disease. The analysis reveals that only the afflicted
individuals share a gene variation for a given complement component. This is suggestive of an
underlying genetic cause or influence. If the function of the gene product is compromised in these
individuals, a correlation between the gene and disease expression is supported. Indeed, it was
found that individuals possessing a defective complement component are highly susceptible to SLE.
It is clear that other genes must be involved in the majority of SLE cases, for most afflicted
individuals do not possess a defective complement system. What other genes might promote SLE?
A number of auto-antigens have been identified and correlated with SLE, implying that an improper
presentation of antigen promotes the aberrant immune response. Two immediate possibilities for
auto-antigen presentation come to mind. Abnormal apoptosis can result in the exposure of
potential auto-antigens to immune cells. Perhaps aberrant copies of genes responsible for the
control of apoptosis can be identified and correlated with autoimmune responses. Again, pedigree
analysis might provide initial evidence for such a link. Another possibility involves the MHC
molecules utilized to present antigens to helper T cells. Variations in these cell surface features
might help promote abnormal immune responses such as SLE. MHC allele variations are a common
target for pedigree analysis, though their abundance and large inherent variation causes great
difficulty in establishing causal relationships.
In this exercise, students are challenged to investigate the genetic influence of a given MHC allele
and a complement allele on the manifestation of SLE. Given a family pedigree, students are directed
to analyze allelic variations among the individuals and to determine if the gene is a potential
candidate for further analysis. Students will simulate PCR generation of allele ‘morphs’ and the
process of gel electrophoresis, followed by a comparison of the DNA results to the presented
pedigree.
Procedure
1. Find the primer sites for the MHC gene on DNA sample #1 (recall that there are two strands for
each sample, because individuals are diploid, possessing homologous maternal and paternal
chromosomes). Remember that there will be primer sites on each strand of a DNA duplex. The
primer sites (5’ – 3’) for the MHC allele are AAAA and CCCC.
2. Starting at the 5’ end of the primer site, write in (use pencil) the appropriate complementary
base (A, C, G, or T) immediately adjacent to each template base. Stop when you have completed
the 5’ end of the opposite primer site. Remember to extend the primers in the 5’-3’ direction,
for this is the only direction that can be catalyzed by a polymerase enzyme. EXAMPLE: Notice
that the top complementary strand (bold) has been completed, whereas the bottom polymer is
in the process of 5’-3’ extension.
5’ggggcagtacaatattcgacgata 3’
3’ atcgatgccccgtcatgttataagctgctatagcacacacacattttgcaag 5’
5’ tagctacggggcagtacaatattcgacgatatcgtgtgtgtgtaaaacgttc 3’
3’ agctgctatagcagacacacatttt
3. Cut the two new polymers for each homologous chromosome and tape them together, taking
care to align complementary bases. These paper samples represent PCR products. Count the
number of base pairs and record this on the back of the taped paper strand.
4. Repeat this procedure for the remaining DNA samples.
5. Cut out the DNA molecular-size markers (standards).
6. Using the teacher-designated flat area, prepare 11 ‘electrophoresis lanes’ (10 DNA samples and 1
standard).
7. Separate the PCR products down the lanes, taking care to align them in accordance with their
approximate positions based on the positions of your standards.
8. Analyze the results by comparing PCR profiles with the provided family pedigree. Determine if
there appear to be any consistencies (correlations) between a given PCR product (allele
morph) and the occurrence of SLE.
9. Repeat the entire procedure for the apoptosis-related gene. The primer sites for this gene (5’ –
3’) are TTTT and GGGG.
SLE Pedigree
5
1
= SLE
9
4
3
6
= Normal
2
7
10
8
Questions
1. What two techniques can permit researchers to obtain a DNA profile (“fingerprint”) from an
individual?
2. What feature of allelic variations (morphs) allows them to be differentiated by gel
electrophoresis?
3. List four ingredients required to perform PCR.
4. Why are two primers required to amplify a single gene?
5. What is the purpose of running a lane of DNA standards during gel electrophoresis?
6. Why are pedigrees valuable in searches of gene correlation for disease? What other populations
might be quite useful?
7. How many different MHC alleles did you find within the test population? How many apoptosis
alleles?
8. In your study, did you find any correlation between allele combinations and the occurrence of
SLE?
9. If this study would have been authentic (not a simulation), would the results be conclusive?
Why or why not?
10. What further studies would you recommend to enhance this investigation?
11. Describe how the techniques simulated in this exercise can be employed in fields such as
criminal forensics.
DNA Results
Number
of Bases
82
80
78
76
74
72
70
68
66
64
62
60
58
54
Sample Sample
Number Number
1
2
Sample Sample
Number Number
3
4
Sample Sample
Number Number
5
6
Sample
Number
7
Sample Sample
Number Number
8
9
Sample
Number
10
DNA 1