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Review
. 2015 Jan;67(1):1-10.
doi: 10.1002/art.38800.

Disease mechanisms in rheumatology--tools and pathways: defining functional genetic variants in autoimmune diseases

Shaofeng Wang  1 , Graham B Wiley, Jennifer A Kelly, Patrick M Gaffney
Affiliations
Free PMC article
Review

Disease mechanisms in rheumatology--tools and pathways: defining functional genetic variants in autoimmune diseases

Shaofeng Wang et al. Arthritis Rheumatol. 2015 Jan.
Free PMC article
No abstract available

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Figures

Figure 1
Figure 1
Characterization of the interaction between DNA and nuclear proteins. A, Electrophoretic mobility shift assay (EMSA) and supershift assay. Lanes contain (from left to right) free 32P-probe, EMSA, EMSA in the presence of specific cold probe, EMSA in the presence of nonspecific probe, EMSA in the presence of antibodies against transcription factors. B, Chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP-qPCR) assay. Antibody against transcription factor is used to precipitate the crosslinked nuclear proteins and the target DNAs. Reverse transcription–qPCR (RT-qPCR) assay is used to determine the amount of precipitated DNA.
Figure 2
Figure 2
Characterization of functional variants using a luciferase reporter gene assay system. A, The promoter/enhancer activity assay system is based on a reporter gene encoding luciferase. Sequences of promoter or enhancer carrying candidate variants are cloned in front of the luciferase gene. B, Sequences of the 3′-untranslated region (3′-UTR) carrying candidate variants are cloned into a plasmid DNA after rabbit β-globin.
Figure 3
Figure 3
Detection of interacting genetic regulatory elements by chromatin conformation capture–quantitative polymerase chain reaction (3C-qPCR) assay. A, Interacting promoter and enhancer are crosslinked and digested with an appropriate restriction enzyme. Intramolecular ligation of crosslinked fragments is performed to generate chimeric DNA, the amount of which is then determined by reverse transcription–qPCR assay. B, Bacterial artificial chromosome (BAC) clone carrying the DNA region of interest is digested with the same enzyme used in C, followed by a random ligation to generate a positive control template containing all possible ligation products. C, The interaction frequencies for anchor/primer sets are shown by the arrows. Arrows marked NFL1–3 indicate nonfunctional looping. The strength of the interaction frequency is proportional to the weight of the arrow. Note that the interaction frequency is inversely related to the genome distance. Interaction resulting from functional looping is shown by the orange arrow. The line graph at the bottom shows the relative crosslinking frequency (RCF).
See this image and copyright information in PMC

References

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