Project Description

Description

Abstract

When your vision changes, your life will change.

Glaucoma is a serious eye disease that can affect anyone. This disease cannot be cured, and the advanced medical technology today can only slow down its progression. If glaucoma is detected late, the start of treatment will also be delayed. Meanwhile, as glaucoma progresses, vision may become blurred, narrowed, or in the worst case scenario, vision may be lost. This can reduce the quality of vision (QOV) and significantly impact daily life.

In the field of synthetic biology, there are noteworthy accomplishments in both basic research and applied research, in a wide range of fields such as production industry, medicine, and environmental conservation. However, much of the research is not known to the general public, being far from implementation for being used at home. Although strict regulations to prevent the spread of genetically modified organisms are necessary as the responsibility of scientists, they indirectly delay social implementation.

POIROT is developed aiming to detect glaucoma specific microRNA at home. To rapidly realize practical application in society, a cell-free system based on enzymes and nucleic acids are adopted, which has fewer regulations for use outside the laboratory. Also, the series of reactions proceeds isothermally, which highlights useability. Moreover, as applications, this system is potentially useful to efficiently amplify and quantify disease-specific (trace amounts) miRNA in body fluids, as a detection for diseases other than glaucoma.

Inspiration

The eyes are one of our most important sensory organs, yet many modern people have problems.
It's true even of people close to us. For example, one of our UTokyo 2024 team members has been diagnosed with pre-glaucoma. Another had suffered from congenital cataracts in the past. Many other members wear glasses or contact lenses, and are more or less worried about their eyes.
We were very interested in glaucoma as our own future risk. This disease, which can even lead to blindness, is very familiar to everyone. In addition to congenital factors, increased intraocular pressure and myopia are also risk factors of the disease.

Of course we are not the only ones who feel anxious about eye diseases. In this era of widespread electronic devices and consequential eyesight declining concerns, eye diseases like glaucoma will become more prevalent. Then, we decided to think about possible approaches to glaucoma from the perspective of synthetic biology.

Initially, we considered glaucoma treatment during project design. Glaucoma treatment has been widely studied, but the technology to cure the disease fundamentally has not yet been established. Through HPs with experts involved in glaucoma research and clinical practice, we found significance in early diagnosis. Early detection and early treatment of glaucoma can reduce the risk of visual field loss and blindness. In Japanese society, where the average life expectancy is increasing, it will lead to maintaining lifelong QOL (Quality of Life) and QOV (Quality of Vision).

OASYS, a diagnostic aid for depression developed by IISER TVM 2023 1, has a wide range of applications in research and clinical settings. Its broad range of uses beyond just a diagnostic tool inspired our detection device and helped us design the project. Its method to use miRNA as a biomarker to detect disease is currently attracting attention in POCT (Point-of-Care Testing). We use tear fluid as a source of glaucoma biomarker miRNA. Tear fluid is an ideal body fluid that can be collected minimally invasively and contains few impurities.

Glaucoma

Glaucoma is a serious eye disease that poses a significant threat to your vision by damaging the optic nerve, which can gradually narrow your visual field. If left untreated, it potentially leads to blindness. The main symptoms include visual field loss, iridescence, eye pain, and decreased visual acuity 2, 3. The number of glaucoma patients is increasing in Japan, and according to a 2020 survey, approximately 2.3 million people are suffering from glaucoma 4. It is particularly common among the elderly, and the number of patients is expected to continue to increase as the aging population. (Figure 1.)

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Figure 1. Trends in the number of glaucoma patients in Japan from 1987 to 2020
Note. The survey was conducted every three years by Japan's Ministry of Health, Labor and Welfare. The figure was created originally, based on the data.

Inside your eyes, a fluid called aqueous humor is carrying nutrients instead of blood, and its pressure, known as intraocular pressure, is maintaining the shape of the eye. The main cause of glaucoma is increased intraocular pressure, and it is classified into primary glaucoma, congenital glaucoma, and secondary glaucoma depending on the factors that cause increased intraocular pressure. Primary glaucoma is further sorted into open-angle glaucoma and angle-closure glaucoma 3.

Primary open-angle glaucoma is a chronic disease with clogged waterproof outlets and increasing intraocular pressure, whose symptoms progress slowly 3. Furthermore, glaucoma can develop even if the intraocular pressure remains in the normal range, which is called normal-tension glaucoma 3. According to the Japanese Glaucoma Society, more than 70% of glaucoma patients in Japan have normal-tension glaucoma 5.

  • Primary obstructive glaucoma, both chronic and acute types, occur when the flow of waterproofing is obstructed due to narrowing angle, leading to increased intraocular pressure 3.
  • Congenital glaucoma is caused by an underdeveloped angle at birth, and is primarily treated by surgery 6.
  • Secondary glaucoma is caused by increased intraocular pressure due to trauma, corneal disease, retinal detachment, eye inflammation, or medications such as steroid hormones 3.

Symptoms

Chronic glaucoma is called a "silent disease" because there are almost no symptoms in the early stages, and the symptoms often progress unnoticed 3. The initial symptom includes a scotoma that appears slightly off the center of the eye, and it is difficult for people to notice something abnormal on their own. In the middle stages, visual field defects become widespread. However, the visual field is compensated for by the other eye, and the abnormality is often not noticed. In the late stage, visual field loss and visual acuity deterioration become severe, interfering with daily life. If left untreated, it can lead to blindness. (Figure 2.) 3

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Figure 2. Examples of visual field for each stage of glaucoma

Glaucoma is a disease that progresses unnoticed and can even lead to blindness. It is reported that it is the leading cause of premature blindness in Japan, accounting for 40.7% of blindness cases 7. Therefore, eye exams are recommended to detect glaucoma earlier. Our Human Practices with glaucoma patients provided an opportunity to learn about the reality of their symptoms.

Currently, treatment cannot restore lost vision, but early detection and appropriate treatment can slow the progression of glaucoma. Treatment methods include drug therapy and surgery to control the amount and flow of aqueous humor 5. Decline in visual ability is directly linked to lifelong decline in QOL and QOV. In Japan, where the average life expectancy continues to increase 8, it is important to detect and treat glaucoma early in order to maintain QOL and QOV for a longer period of time.

POIROT for Glaucoma

Glaucoma is diagnosed at a hospital by performing an intraocular pressure test, fundus examination, and visual field test 9. However, since early symptoms are very much difficult to be noticed, the disease has often progressed by the time patients recognize the need for testing. POIROT is a detection device suitable for simple in-home detection for glaucoma. People who have no symptoms and feel no urgency to go to the hospital, or people who are busy with work or other reasons and don't have time to go to the hospital, can easily use it for a short time and at low cost. It is expected that it will make glaucoma more familiar to people and will be useful for early detection and treatment.

Our Solution

Our system, POIROT, starts with glaucoma biomarker miRNA in tear fluid. MiRNAs are a family of non-coding RNAs of about 18-22 nt, and function as important regulators of gene expression 10. Being released outside cells, they carry information about physiological and pathological processes. In other words, miRNAs in biological fluids can be used as biomarkers for detection 11.

POIROT detects glaucoma by amplifying and quantifying the signal from glaucoma biomarker miRNA as a starting material. It was designed with usability as its first priority, with the goal of being easy to use at home.

Therefore, the following features are emphasized:

  • Uses tear fluid, a body fluid that can be collected minimally invasive
  • All processes proceed isothermally
  • No special equipment is needed

The concentration of miRNA in tear fluid varies depending on the type, but it can be reasonably estimated to be around several fmol/L 12. Femto is a prefix that stands for \(10^{-15}\) 13. Tear fluid contains higher concentrations of miRNA compared to plasma and urine 12, but have not received much research attention, and progress has been limited. Any biomarker miRNAs have not been reported that are specifically present or concentrated in glaucoma. Here is the result of our investigation on miRNAs related to glaucoma.

  • miR-10b-5p: miR-10b-5p is a miRNA involved in neurodegeneration. It has been suggested that miRNAs related to neurodegeneration are contained in tear fluid 14.
  • miR-375: Overexpression of miR-375 can reduce brain-derived neurotrophic factor (BDNF)-induced increase in neurite outgrowth 15. Decreased BDNF in glaucoma negatively impacts retinal neuroprotection 16.
  • miR-30b-5p: Shown to promote neurite outgrowth of retinal ganglion cells 17.

These three miRNAs are contained in aqueous humor, and it has been reported that their expression levels differ by about 100-fold between healthy subjects and glaucoma subjects 18. These are promising candidates for glaucoma biomarker miRNAs, and are expected to be contained in tear fluid as well, although research has not yet been conducted.

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Figure 3. Amplification of miRNA in tear fluid

Biomarker miRNA is amplified as DNA by isothermal amplification using a DNA polymerase 19with strand displacement activity 20. The amplified DNA activates the E.coliCas3-E.coliCascade/crRNA complex with collateral activity, which has the ability to indiscriminately cleave the surrounding single-stranded DNA 21. The activated Cas3-Cascade/crRNA complex amplifies the intensity of the output signal by cleaving reporter molecules which include ssDNA. The output is determined by the color reaction of the Lateral Flow Assay (LFA). Two calibration curves appear if glaucoma for positive, while one calibration curve appears if negative 22.

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Figure 4. POIROT

POIROT is not limited to glaucoma detection at home. It has high flexibility in that any miRNA can be amplified and quantified by simply changing the sequence of a part of the nucleic acid used for amplification. Therefore, with a disease-specific biomarker miRNA, POIROT can be used in a wide range of applications other than glaucoma. For example, it can be used for diabetic retinopathy, whose biomarker miRNA has been reported in tear fluid 23. In addition, with its high sensitivity, it is highly likely to be applied to body fluids other than tear fluid. For instance, POIROT can contribute to the early detection of malignant neoplasms such as pancreatic cancer, and chronic diseases with few subjective symptoms, that are difficult to detect early though desired.

Amplification System

Research for detecting trace amounts of miRNA is progressing, and improvements are being made. Examples in past iGEM projects include aptamers 1, 24, Toehold-Mediated Strand Replacement (TMSD) 25, Strand Displacement Amplification (SDA) 26, Rolling Circle Amplification (RCA) 26, 27, and Loop-mediated Isothermal Amplification (LAMP) 27, 28. However, these systems are far from practical use at home in that their sensitivity and specificity are insufficient, or they require some advanced equipment to detect fM order miRNA.

To determine the appropriate method for detecting miRNAs, various amplification systems were investigated, from both Wet Lab and Dry Lab viewpoints.
Click here for more expamination:

As a result, the amplification system in POIROT was decided to be a connected series of Three Way Junction-SDA (TWJ-SDA) 29, Multistep-SDA 30, CRISPR-Cas3, and LFA 22.

Amplification

TWJ-SDA

If three nucleic acids have complementary parts to each other, they can form a TWJ complex. The TWJ complex has higher sequence specificity than a complex of two complementary nucleic acids, and may reduce false positives. In principle, it is possible to design a TWJ complex specific to any sequence, making it highly applicable.

Multistep-SDA

SDA is a method to generate large amounts of ssDNA by using DNA polymerase which shows strand displacement activity and a nickase that recognizes double-stranded DNA and inserts a nick into one of the strands. There are numbers of variations: the target, amplicon, and reaction temperature can be changed according to the purpose. We focused on "L-TEAM", an amplification system developed by Komiya 30. "L-TEAM" is a system that combines the simplest SDA reaction through two or three steps, and has the characteristic of amplifying approximately 24 mer ssDNA. SDA reaction originally has a problem of nonspecific amplification, where amplification occurs even in the negative control, but "L-TEAM" suppresses the negative control by various measures. With reference to "L-TEAM", we have investigated various measures to suppress the negative control and have devised our own "Multistep-SDA".

Cas3-Cascade/crRNA complex

Cas3-Cascade/crRNA complex is a protein complex consisting of Cas3 and the Cascade-crRNA complex. CrRNA, which functions as a sequence-specific guide molecule, binds to the Cascade complex to recognize dsDNA. The Cas3 protein is a bifunctional enzyme that has both 3' to 5' ATP-dependent helicase activity and nuclease activity that mainly targets ssDNA.

The Cas3-Cascade/crRNA complex activated by dsDNA induces large-scale DNA cleavage extending to the surrounding region of the sequence specified by the crRNA. This property is mainly applied to genome reduction and removal of unnecessary DNA regions 31.
On the other hand, the activated Cas3-Cascade/crRNA complex also has the function of indiscriminately cleaving surrounding ssDNA through collateral activity. In our project, this activity achieves a different purpose than genetic modification. Specifically, we aim to induce cleavage of ssDNA contained in reporter molecules present in large quantities in the surrounding area.

Detection

Lateral Flow Assay

The input signal, starting from miRNA, is converted, first to dsDNA by TWJ-SDA and Multistep-SDA. Finally, the signal is converted from dsDNA to color and visualized so that anyone can easily judge the results. Therefore, we focused on LFA, which is used in many methods to present the results such as new influenza and pregnancy tests. This method is familiar to people because the results are visually easy to understand and it is widely used for COVID-19 detecting and other purposes. In addition, it shows high performance with the Cas3-Cascade/crRNA complex which has a long recognition sequence and high specificity. We designed our system to be user-friendly and accurate: the result can be judged by the color of the standard curve by utilizing the lateral activity of LFA and Cas3-Cascade/crRNA complex.

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Figure 5. LFA strip when positive

The types and characteristics of the molecules used in our system

  • Biotin-ssDNA-FITC: Reporter molecule. Its ssDNA portion is cleaved by Cas3-Cascade/crRNA complex.
  • AuNP-Antibody: Causes an antigen-antibody reaction with FITC of reporter molecules and binds strongly. AuNPs show a reddish color in general, varying depending on particle size.
  • Streptavidin: Binds strongly to reporter molecules Biotin.
  • Secondary antibody: Binds strongly to antibody.

Being positive for glaucoma means there is sufficient amplified dsDNA. It activates the Cas3-Cascade/crRNA complex, and indiscriminate ssDNA cleavage due to collateral activation occurs. A part of the reporter molecule is cleaved and separated into biotin and FITC, which goes through the flow path and then binds strongly to the AuNP-antibody through an antigen-antibody reaction. Biotin or the reporter molecule binds strongly to the streptavidin on the control line, and the FITC-antibody-AuNP binds strongly to the secondary antibody on the test line. This causes both the control line and the test line to turn red.

On the other hand, being negative for glaucoma means there is no amplified dsDNA. Consequently, Cas3-Cascade/crRNA complex is not activated, and indiscriminate ssDNA cleavage due to collateral activation does not occur. The reporter molecule goes through the flow path and binds strongly to the AuNP-antibody through an antigen-antibody reaction. Almost all reporter molecules bind strongly to streptavidin on the control line, and cannot bind to the secondary antibody on the test line. In this way, only the control line turns red.

Our System

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Figure 6. Overview of POIROT

  • Tear fluid sample
  • Template0 and helper designed for target miRNA
  • Template1, template2, template3, and ds-template independent of target miRNA
  • DNA polymerase (Bst DNA polymerase, Large fragment)
  • Nickase (Nb.BbvCI)
  • Cas3-Cascade/crRNA complex
  • FITC-ssDNA-Biotin
  • LFA strip

  1. Target miRNA, helper DNA, and template DNA form a TWJ complex in a sequence-specific manner.
  2. DNA polymerase extends DNA from the 5' end of helper DNA.
  3. The extending strand contains a nickase recognition site, where the nickase nicks in a sequence-specific manner.
  4. Polymerase with strand displacement activity extends DNA from the nick, while the short DNA in front (primer0) is peeled off.
  5. Steps 2-4 are repeated, and many primer0s are generated.
  6. Primer0 hybridizes to template1, and polymerase extends DNA from the 5' end.
  7. Steps 3, 4, and 6 are repeated, and primer1, primer2, and primer3 are generated one after another.
  8. Primer3 hybridizes to ds-template, and DNA polymerase extends DNA from the 5' end.
  9. The resulting dsDNA is recognized by the Cas3-Cascade/crRNA complex, activating the collateral activity of Cas3.
  10. Collateral cleavage by Cas3 cleaves the FITC-ssDNA-Biotin reporter.
  11. The cleaved reporter molecule flows along the LFA strip.

POIROT

Here is an explanation of how to use POIROT. First, a user collects their own tear fluid using Schirmer strip 32. Second, the collected tear fluid are mixed with the reaction solution and incubated in the device at 37°C for 40 minutes. Then, the mixture and developer are dropped onto the sample pad of the LFA. Finally, the mixture flows over the LFA and reaches the calibration line. Two calibration lines (Control Line and Test Line) turning red means positive, while only one calibration line (Control Line) turning red means negative.
Click here for more detailed experiment information:

Conclusion

POIROT is a glaucoma detection device that amplifies and quantifies glaucoma biomarker miRNA in tear fluid. POIROT is composed of a cell-free enzyme and nucleic acid-based amplification system and a lateral flow coloring system. It is optimized for home use as a diagnostic aid for glaucoma so that it can specifically detect trace amounts of miRNA without the use of advanced equipment. Early detection is only the first step towards solving problems on glaucoma. The next step is to treat it early and solve the problem fundamentally. POIROT is a big step forward that provides a new vision for our future.

Future

POIROT has great flexibility. It is expected that almost all miRNAs can be detected by POIROT with minimal sequence changes. Therefore, with a disease-specific biomarker miRNA, POIROT can be used in a wide range of applications other than glaucoma. For example, POIROT can contribute to the early detection of malignant neoplasms such as pancreatic cancer and Alzheimer's Syndrome, that are difficult to detect early though desired. POIROT has a wide range of applications beyond glaucoma, and will bring about great progress in earlier detection of diseases.

References

  1. IISER TVM 2023. https://2023.igem.wiki/iiser-tvm/home

  2. KoKyo zaidan hojin tyoju kagaku shinko zaidan. (2022). Ryokunaisyo no syojo. https://www.tyojyu.or.jp/net/byouki/ryokunaishou/shoujou.html

  3. Iryo hojin syadan kaiya. Ryokunaisyo. https://www.kaiya-eyes.com/treatment/disease/glaucomal

  4. Kousei roudousho. (2020). Reiwa 2nen kanja tyosa syobyo bunruihen (syobyobetsu nenzi suiihyo). https://www.mhlw.go.jp/toukei/saikin/hw/kanja/10syoubyo/dl/r02syobyo.pdf

  5. Nihon ganka gakkai. Ryokunaisyo no tiryo. https://www.nichigan.or.jp/public/disease/treatment/item05.html

  6. Nihon ryokunaisho gakkai. Japanese Glaucoma Society Tajimi Glaucoma Epidemiology Survey. https://www.ryokunaisho.jp/general/ekigaku/tajimi.php

  7. Matoba, R., Morimoto, N., Kawasaki, R., et al. (2023). A nationwide survey of newly certified visually impaired individuals in Japan for the fiscal year 2019: Impact of the revision of criteria for visual impairment certification. Japanese Journal of Ophthalmology, 67(4), 346-352. https://doi.org/10.1007/s10384-023-00986-9

  8. Ministry of Health, Labor and Welfare. (2020). Overview of life tables by prefecture in 2020. https://www.mhlw.go.jp/toukei/saikin/hw/life/tdfk20/dl/tdfk20-10.pdf

  9. Japan Ophthalmologists Association. Yoku wakaru ryokunaisyo -tiryo to shindan-. https://www.gankaikai.or.jp/health/49/index.html

  10. Hammond, S. M. (2015). An overview of microRNAs. Advanced drug delivery reviews, 87, 3-14. https://www.sciencedirect.com/science/article/pii/S0169409X15000940

  11. O'Brien, J., Hayder, H., Zayed, Y., & Peng, C. (2018). Overview of microRNA biogenesis, mechanisms of actions, and circulation. Frontiers in endocrinology, 9, 402. https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2018.00402/full

  12. Weber, J. A., Baxter, D. H., Zhang, S., Huang, D. Y., Huang, K. H., Lee, M. J., Galas, D. J., & Wang, K. (2010). The MicroRNA spectrum in 12 body fluids. Clinical Chemistry, 56(11), 1733-1741. https://doi.org/10.1373/clinchem.2010.147405

  13. The NIST Reference on Constants, Units, and Uncertainty. https://physics.nist.gov/cgi-bin/cuu/Value?rp

  14. Raga-Cervera, J., Bolarin, J. M., Millan, J. M., Garcia-Medina, J. J., Pedrola, L., Abellán-Abenza, J., Valero-Vello, M., Sanz-González, S. M., O'Connor, J. E., Galarreta-Mira, D., et al. (2021). miRNAs and genes involved in the interplay between ocular hypertension and primary open-angle glaucoma. Oxidative stress, inflammation, and apoptosis networks. Journal of Clinical Medicine, 10(11), 2227. https://doi.org/10.3390/jcm10112227

  15. Liu, Y., Wang, Q., Wen, J., Wu, Y., & Man, C. (2021). MiR-375: A novel multifunctional regulator. Life Sciences, 275, 119323. https://doi.org/10.1016/j.lfs.2021.119323

  16. Nishijima, E., Honda, S., Kitamura, Y., Namekata, K., Kimura, A., Guo, X., ... & Harada, T. (2023). Vision protection and robust axon regeneration in glaucoma models by membrane-associated Trk receptors. Molecular Therapy, 31(3), 810-824. https://doi.org/10.1016/j.ymthe.2022.11.018

  17. Han, F., Huo, Y., Huang, C. J., Chen, C. L., & Ye, J. (2015). MicroRNA-30b promotes axon outgrowth of retinal ganglion cells by inhibiting Semaphorin3A expression. Brain Research, 1611, 65-73. https://doi.org/10.1016/j.brainres.2015.03.014

  18. Cho, H. K., Seong, H., Kee, C., et al. (2022). MicroRNA profiles in aqueous humor between pseudoexfoliation glaucoma and normal tension glaucoma patients in a Korean population. Scientific Reports, 12, 6217. https://doi.org/10.1038/s41598-022-09572-4

  19. NEB. DNA Polymerase Selection Chart. https://www.neb.com/ja-jp/tools-and-resources/selection-charts/dna-polymerase-selection-chart

  20. Yan, L., Zhou, J., Zheng, Y., Gamson, A. S., Roembke, B. T., Nakayama, S., & Sintim, H. O. (2014). Isothermal amplified detection of DNA and RNA. Molecular BioSystems, 10(5), 970-1003. https://doi.org/10.1039/C3MB70304E

  21. Yoshimi, K., Takeshita, K., Kodera, N., et al. (2022). Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3. Nature Communications, 13, 4917. https://doi.org/10.1038/s41467-022-32618-0

  22. Yoshimi, K., Takeshita, K., Yamayoshi, S., Shibumura, S., Yamauchi, Y., Yamamoto, M., Yotsuyanagi, H., Kawaoka, Y., & Mashimo, T. (2022). CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus.iScience 25, 103830, 1-13. https://doi.org/10.1016/j.isci.2022.103830

  23. Hu, L., Zhang, T., Ma, H., Pan, Y., Wang, S., Liu, X., ... & Liu, F. (2022). Discovering the secret of diseases by incorporated tear exosomes analysis via rapid-isolation system: iTEARS. ACS Nano, 16(8), 11720-11732. https://doi.org/10.1021/acsnano.2c02531

  24. IIT ROORKEE 2023. https://2023.igem.wiki/iit-roorkee/home

  25. PATRAS MEDICINE 2022. https://2022.igem.wiki/patras-medicine/

  26. NEFU CHINA 2021. https://2021.igem.org/Team:NEFU_China

  27. LINKOPING SWEDEN 2022. https://2022.igem.wiki/linkoping-sweden/index.html

  28. MICHIGAN 2023. https://2023.igem.wiki/michigan/index.html

  29. Ying, X., Yu, W., Su, Liu., Jinghua, Y., Hongzhi, W., Yuna, G., & Jiadong, H. (2016). Ultrasensitive and rapid detection of miRNA with three-way junction structure-based trigger-assisted exponential enzymatic amplification. Biosensors and Bioelectronics, 81, 236-241. https://doi.org/10.1016/j.bios.2016.02.034

  30. Komiya, K., Noda, C. & Yamamura, M. (2024). Characterization of Cascaded DNA Generation Reaction for Amplifying DNA Signal.New Gener. Comput. 42, 237-252. https://doi.org/10.1007/s00354-024-00249-2

  31. Sinkunas, T., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., & Siksnys, V. (2011). Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. The EMBO journal, 30(7), 1335-1342. https://doi.org/10.1038/emboj.2011.41

  32. Schirmer Test. https://www.ncbi.nlm.nih.gov/books/NBK559159/

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