{"id":5839,"date":"2020-08-26T09:47:29","date_gmt":"2020-08-26T09:47:29","guid":{"rendered":"https:\/\/clinlabint.3wstaging.nl\/chip-based-capillary-electrophoresis-for-rapid-diagnosis-of-genetic-disorders\/"},"modified":"2021-01-08T11:39:15","modified_gmt":"2021-01-08T11:39:15","slug":"chip-based-capillary-electrophoresis-for-rapid-diagnosis-of-genetic-disorders","status":"publish","type":"post","link":"https:\/\/clinlabint.com\/chip-based-capillary-electrophoresis-for-rapid-diagnosis-of-genetic-disorders\/","title":{"rendered":"Chip-based capillary electrophoresis for rapid diagnosis of genetic disorders"},"content":{"rendered":"
Prenatal screening and diagnosis is important for early detection and therapy of neonates affected by genetic disorders. Chip-based capillary electrophoresis can analyse small sample volumes quickly and easily. This technique may be used for prenatal as well as pre-implantation genetic diagnosis, where sample volumes are tiny and difficult to obtain. It could resolve many difficulties in current testing methods, which are slow and require large sample volumes.<\/p>\n by Dr Hua Hu and Dr Zhiqing Liang<\/strong><\/p>\n Potential of chip-based capillary electrophoresis for rapid diagnosis of genetic disorders<\/strong> Based on the common capillary electrophoresis, chip-based capillary electrophoresis uses \u2018lab-on-a-chip\u2019 technology. It can implement the sample introduction, reaction, separation and detection, and is a multifunctional, rapid, high performance, low volume, miniature analytical apparatus. The apparatus is not only low volume but also the automatic mode of operation reduces detection time of detection and experimental artefacts. This technique may be used for prenatal and pre-implantation genetic diagnosis where samples are tiny and difficult to obtain. Moreover, the rapid detection time can reduce parent anxiety. <\/p>\n We have optimised the detection beam path, separation gel and laser device of our chip-based capillary electrophoresis system in order to increase the sensitivity, resolution and stability. The improvement of the chip-based electrophoresis detector over the conventional confocal set-up was in the use of a holed reflecting mirror instead of a dichroic mirror, which significantly eliminated the effect of reflected laser light on the fluorescence detection. Moreover, the lasers were focused onto a very small spot (5 μm diameter) giving higher intensity light, thus giving higher efficiency fluorescence excitation. Additionally, the focused spot was much smaller than the width of the separation channel (60 μm), so avoiding the illumination of rough capillary side walls which can cause scattering of laser light, therefore increasing sensitivity. To further facilitate the optical alignment, a CCD imaging system was installed above the poly (methyl methacrylate) (PMMA) chip, confocal with the laser-induced fluorescence (LIF) detector. Also a dynamic movie of the electrophoretic process could be recorded using the CCD imaging system [Figure 1]. The device exhibited good reproducibility and is suitable for high-throughput applications.<\/p>\n We employed dual channel sample detection, with the two different wavelengths, for example Cy3 and Cy5. A standard substance was labelled with the fluorescent Cy3 label and the test sample was labelled the Cy5 fluorophore. By comparing the fluorescence of the Cy3 and Cy5 samples, we can deduce the size of the test sample and ascertain the genotype [Figure 2]. Our results show that chip-based capillary electrophoresis is an accurate, rapid and highly sensitive detection method suitable for prenatal diagnosis. Chip-based capillary electrophoresis may be promising for the prenatal and pre-implantation genetic diagnosis of β-thalassemia, where high sensitivity is vital as sample volume is limited and difficult to obtain. Sensitivity studies showed the chip-based capillary electrophoresis system was capable of detecting 1 ng of genomic DNA (1 ng\/μl), and had a linear range of detection of 1\u201350 ng\/μl. The detection system requires only 1 μl of sample at 0.04 nM. We utilised chip-based capillary electrophoresis and developed rapid assays for prenatal diagnosis of β-thalassemia. Chip-based capillary electrophoresis decreased the analysis time for genotyping to 200 s. The separation time is shorter than gel electrophoresis and capillary electrophoresis, which accelerates the prenatal diagnosis. Moreover we detected the size ladder and samples simultaneously by a dual-channel detection system, which are labelled with Cy3 and Cy5 fluorescence, respectively, to improve the detection precision.<\/p>\n We compared three methods including agarose gel, polyacrylamide gel and chip-based capillary electrophoresis. Traditional agarose gel electrophoresis and ethidium bromide staining is cheap but is potentially harmful to health, and the sieving capability of agarose gel is limited to 50 bp, which makes it difficult to separate multiple PCR products. Polyacrylamide gel has a higher separation capability but is time-consuming. Chip-based capillary electrophoresis is faster and can discriminate 4\u201310bp differences, something that is not possible using traditional gel electrophoresis [1], making it convenient for primer design. Reverse dot blotting is easy and quick but requires large amounts of DNA (about 2 μl of 0.1 μg\/μl DNA), and the process is cumbersome, requiring more than 7 hours. Chip-based capillary electrophoresis is sensitive and only requires about 1ng\/μl DNA which is important for precious sample detection. Only 3.5 hours are needed, including the procedure of extracting DNA, PCR, purification and detection, which reduces the time for awaiting the result and eases the anxiety of patients. It allows 10 bp resolution and takes only a few seconds. These results show that chip-based capillary electrophoresis is a quick, sensitive detection system with improved resolution. The sensitive characteristics of chip-based capillary electrophoresis provide obvious advantages over slab-gel electrophoresis and capillary electrophoresis for biomedical and clinical applications.<\/p>\n Although chip-based capillary electrophoresis has tremendous potential in disease diagnostics, its wider use has been limited by the size and cost of the instrumentation. Most reports of chip-based capillary electrophoresis have used glass or silica as the base materials for chip fabrication. We utilised the polymer substrate PMMA which made the chips less expensive and easier to make. The cost of analysis of one sample was about 10$ (75\u00a5), which is identical to reverse dot blotting and acceptable to most patients. There are chip-based capillary electrophoresis designed with integrated circuit chips which include inexpensive portable systems, complementary metal-oxide-semiconductor chips and low-cost components [2]. This instrument is powered and controlled using a universal serial bus interface to a laptop computer which can readily analyse the DNA produced by a standard medical diagnostic protocol. The improvement of chip-based capillary electrophoresis will facilitate its wide use in prenatal diagnosis.<\/p>\n Possible future applications<\/strong> The discrimination of single-nucleotide difference between circulating DNA samples is technically challenging and demands the adoption of highly sensitive and specific analytical systems. Chip-based capillary electrophoresis can analyse small samples speedily and conveniently. We constructed the platform of chip-based capillary electrophoresis to detect the point mutations and achieved prenatal diagnosis of β-thalassemia quickly by detecting fetal DNA in maternal plasma. This chip-based capillary electrophoresis detection system is capable of the non-invasive prenatal diagnosis of β-thalassemia. Its use would facilitate prenatal diagnosis of the genetic disorder rapidly and sensitively. This method has high sensitivity, high-speed and high throughputs, and is very suited for prenatal diagnosis.<\/p>\n Most genetic diseases are a result of point-mutations and DNA fragment deletions. The main DNA defect of β-thalassemia is a point-mutation. However, the gene defects of α-thalassemia include point-mutations and klenow fragment deletions. In the clinic, point-mutations are generally detected by reverse dot blotting or sequencing, and the deletion detected by Gap-PCR and agarose gel electrophoresis [6]. These techniques often require two different detection methods and equipment, and are labour intensive and time-consuming. Combining chip-based capillary electrophoresis with multiplex allele specific PCR, results in the sensitive and reliable detection of the point mutation. This method can also be used to separate and detect the PCR products of different length arising due to deletion events. Therefore, using chip-based capillary electrophoresis, we may detect not only point-mutations but also deletions, and so can simultaneously detect α-thalassemia and β-thalassemia, which is rapid and convenient. Conclusion<\/strong> References<\/strong> The authors<\/strong> *Corresponding author: Prenatal screening and diagnosis is important for early detection and therapy of neonates affected by genetic disorders. Chip-based capillary electrophoresis can analyse small sample volumes quickly and easily. This technique may be used for prenatal as well as pre-implantation genetic diagnosis, where sample volumes are tiny and difficult to obtain. It could resolve many difficulties […]<\/p>\n","protected":false},"author":2,"featured_media":9618,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[52],"tags":[],"_links":{"self":[{"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/posts\/5839"}],"collection":[{"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/comments?post=5839"}],"version-history":[{"count":0,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/posts\/5839\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/media\/9618"}],"wp:attachment":[{"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/media?parent=5839"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/categories?post=5839"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/clinlabint.com\/wp-json\/wp\/v2\/tags?post=5839"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
Genetic diseases often lead to disability or mortality. As they are difficult to cure, prenatal screening and diagnosis is important. Many techniques are used to detect such disorders, such as gel electrophoresis, reverse dot blot methodology, allele-specific oligonucleotide probes (ASO), real-time PCR, mass-spectrometry (MS), sequencing and so on. These methods are accurate for determining the gene mutation, but have low sensitivity and are time-consuming.<\/p>\n
The advantages and limitations of this technique for prenatal diagnosis of β-thalassemia<\/strong>
Thalassemia is the commonest genetic disorder worldwide, especially in the Mediterranean and Asia. Severe thalassemia is associated with high mortality. Prenatal screening for thalassemia is important to prevent the spread of the condition and to give at-risk couples the option of avoiding an affected child. Thus the prenatal screening of the carrier parent and affected fetus is strongly recommended in developing countries where the treatment of affected patients is expensive and may not available.<\/p>\n
Most genetic diseases are diagnosed by invasive prenatal testing, which carries a high risk of abortion, infection and other complications. Fetal cells and cell free fetal DNA [3] in the maternal circulation were discovered nine years ago and can be used for non-invasive prenatal fetal sex determination, and diagnosis of chromosome 21 trisomy and RhD. At present targeted massively parallel sequencing of maternal plasma is used for non-invasive prenatal diagnosis of β-thalassemia [4]. However, it is not applied widely, partly because of sample rarity and technological complexity. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted DNA, the analytical sensitivity, and the specificity. Maternal plasma includes maternal and fetal DNA of which the fraction ranges from a few percent or lower early in pregnancy and increases with gestational age [5]. Hence non-invasive prenatal testing of the fetal genome generally takes place after at least 13 weeks of pregnancy. In addition, some cases require re-testing because of too low fetal DNA content in the first blood sample.<\/p>\n
\n<\/strong><\/p>\n
In future we may use the detection system for non-invasive prenatal analysis of circulating DNA and pre-implantation genetic diagnosis. Moreover, the system could also be used for the detection of α-thalassemia and β-thalassemia together. This could resolve many problems associated with traditional methods of genetic analysis, which are slow and require larger sample volumes and so are not suited for prenatal diagnosis.<\/p>\n
1. Tabe Y, Kawase Y, Miyake K, Satoh N, Aritaka N, Isobe Y, et al. Identification of Bcl-2\/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis. Clin Chem Lab Med 2011; 49(5): 809\u2013815.
2. Behnam M, Kaigala GV, Khorasani M, Martel S, Elliott DG, Backhouse CJ, et al. Integrated circuit-based instrumentation for microchip capillary electrophoresis. IET Nanobiotechnol 2010; 4(3): 91\u2013101.
3. Y M Dennis Lo, Noemi Corbetta, Paul F, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485\u2013487.
4. Lam KW, Jiang P, Liao GJ, Chan KC, Leung TY, Chiu RW, Lo YM. Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: application to β thalassemia. Clin Chem 2012; 15.
5. Palomaki GE, Deciu C, Kloza EM, et al. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study. Genet. Med 2012; 14: 296\u2013305.
6. Rosnah B, Rosline H, Zaidah AW, Noor Haslina MN, Marini R, Shafini MY, et al. Detection of common deletional alpha-thalassemia spectrum by molecular technique in Kelantan, Northeastern Malaysia. ISRN Hematol. 2012; 462969.<\/p>\n
Hua Hu PhD and Zhiqing Liang PhD*
Departments of Obstetrics and Gynecology
Southwest Hospital
Third Military Medical University,
Chongqing, China 400038<\/p>\n
e-mail: zhiq.lzliang@gmail.com<\/em><\/p>\n","protected":false},"excerpt":{"rendered":"