Part:BBa_K4897005
Worm L-RCA (BS DNA 4.0) for detecting Myotonic Dystrophy
What is it?
Worm L-RCA is a novel detection technique based on our previous development of parts detecting P. acne. Our collaborators were very informative and interested in our research in L-RCA. After they successfully replicated our results, they trusted our quality and creativity and asked us for novel detection or diagnosis of certain human diseases. One of the diseases is called Myotonic Dystrophy which is hard to diagnose in the current stage. Here is the general description. Myotonic Dystrophy has different types according to its severity which is based on the amount of repetitions of 5’ CTG 3’[1]. Myotonic Dystrophy Type 1 (DM1) is more severe and has more repetitions than Myotonic Dystrophy Type 2 (DM2). Due to the variability of the disease gene, the current method is flawed in its accuracy and time duration. However, Worm L-RCA is able to solve this issue. It has two DNA fragments working: BS Worm DNA-300 and BS Worm DNA-12. BS Worm DNA-300 is designed and is similar to previous DNAs which bind to P. acne’s DNA. It has the following composition: binding region, amplification region, and random region. The BS Worm DNA-12 is in charge of binding to variable 5' CTG 3' repetitions in the middle of the gene sequence.
Usage and Biology
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| Fig. 1. Worm L-RCA Concept Diagram |
The binding regions, marked in red line, are located on the two sides of CTG repeats. The primer sequences have been designed based on prior research concerning this specific target gene [2]. In contrast to previous DNA binding regions, in the case of Worm L-RCA, the two ends of the binding region do not come together closely when they attach to the target DNA. Instead, the two ends of BS Worm DNA-300 correspond to the two ends of CTG repeats. Inside these DNA fragments, BS Worm DNA-12, comprising four triplets of 3' GAC and 5' CTG repeats in the disease gene, can attach. Subsequently, multiple copies of BS Worm DNA-12 can be ligated together and connected to BS Worm DNA-300. Ultimately, this circular DNA structure can be amplified and examined to predict the presence and severity of Myotonic Dystrophy by determining the number of GAC repetitions by using Sanger Sequencing post-gel electrophoresis. If a patient is afflicted with DM1, the CTG repeats will be considerably longer than that of a typical person. Consequently, a greater number of GAC small fragment primers will attach to the circular DNA. We can then compare the patient's DNA repeat with our reference DNA template, which includes sequences with 30 CTG repetitions, 50 CTG repetitions, 100 CTG repetitions, 200 CTG repetitions, 500 CTG repetitions, and ultimately 1000 CTG repetitions.
Characterization
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| Fig. 2. real time L-RCA for DM1 DNA copies. |
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 218
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 218
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 218
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 218
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 255
Illegal BsaI.rc site found at 13
References
[1]Warner, J. P., Barron, L., Goudie, D., Kelly, K., Dow, D., Fitzpatrick, D. R., & Brock, D. (1996). A general method for the detection of large CAG repeat expansions by fluorescent PCR. Journal of medical genetics, 33(12), 1022-1026
[2]Ikeda, M., Taniguchi-Ikeda, M., Kato, T., Shinkai, Y., Tanaka, S., Hagiwara, H., … Saito, F. (2020). Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach. Molecular Therapy - Methods & Clinical Development, 18, 131–144. https://doi.org/10.1016/j.omtm.2020.05.024
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