Fluorescent genotyping screening for tiny subtelomeric rearrangement assay
Our results strongly support the notion that microtelomeric rearrangements play a very important role in spontaneous mental retardation (approximately 12% in our experiments) and demonstrate that fluorescent genotyping is a very sensitive method for detecting microtelomeric rearrangements. In addition, multifaceted automated genotyping has advantages in terms of quantification and objectivity of results over cytogenetic analyses, which require more technical experience and high variability of results; genotyping also has the advantage of allowing direct identification of the biparental origin of the rearranged chromosomes. Finally, microsatellite DNA technology is uniquely suited for the discovery of uniparental diploids associated with several human disease mechanisms. Source: Molecular Cytogenetics Technology and Applications.
Operation method
Fluorescent genotyping screening for tiny subtelomeric rearrangement assay
Materials and Instruments
Sample DNA Move I. PCR amplification of telomeric satellites 1. Melt the primers on ice and mix gently, centrifuge slightly. 2. For each marker, prepare a mixture of 12 reactions consisting of 196 μL of autoclaved distilled water, 32.5 μL of 10XPCR Buffer, 9.75 μL of Chlorase, 32.5 μL of dNTP mixture, 2uL each of forward and reverse primers, and 1U of Taq enzyme, and manipulate and store on ice. 3. Add 15uL of the above PCR reaction mix to 10uL of DNA and seal the 96-well plate with aluminum foil caps. 4. Before amplification, place the 96-well plate on a thermal cycler and denature at 94°C for 5 min. 5. Cycle at 94°C for 30 s, 55°C for 30 s and 72°C for 45 s for 35 cycles, with a final extension time of 72°C for 7 min. Samples can be stored at -20°C for later use. 1. Gel electrophoresis: Take 3uL of sampling buffer and mix it with 5uL of PCR products, and put the sample onto a 1.5% agarose gel containing 5mg/ml EB prepared with 1XTBE. 2. Electrophoresis at 100V for 30min using appropriate molecular quality standards. 3. Transfer the electrophoresed gel to a UV reflectometer to observe the DNA and ensure the correct amplification of microsatellites. 1. Place the glass plates and spacers in the designated box according to the method listed in the ABI Automated Sequencer manual. 2. Mix 3 mL of LongRanger gel solution with 3 mL of 10X TBE, add 10.8 g of urea, dissolve, add deionized water to 30 mL, and filter through a 0.45um membrane. 3. Add 150 μL of 10% APS and 21 μL of TBE and pour the gel to polymerize for 1 hour. 4. Load the gel cassette into the sequencer according to the instructions. 5. Mix equal amounts of each amplification product together after PCR amplification as shown in Table 2 (see Note 3). 6. Freshly prepare the Sampling Mix: 0.5 μL of GS-400 HDROX, 1xL of Sampling Buffer and 5 ml of deionized formamide. 7. Mix 2 μL of PCR product mix with 3 pL of sample mix. 8. Denature at 90°C for 3 min, then quickly place on ice. 9. Spot the sample into the glue hole. 10. The application runs the GS36D2400 module to collect data. 11. Analyze the data using GeneScan software as described in the product manual. 12. Determine allele size using GenoTyper software. 1. Studies have shown that cytogenetically unrecognizable unbalanced translocations are responsible for intellectual disability in CT thalassemia mental retardation syndrome (ATR-16 [16]), Wolf-Hirchhorn syndrome [17], Miller-Diecker syndrome [18], and cri-du-chats syndrome [19]. Causes. 2. A problem that may arise with this approach is how to determine the phenotype caused by telomere rearrangements. This relationship is clear when there is more than one affected offspring and telomere rearrangement is co-segregating with mental retardation. In the absence of a family history of mental retardation, evidence of a biological effect of telomere rearrangement can be obtained when the rearrangement involves a region that was missing in a previous case of mental retardation, or when the missing fragments are very large. In other cases, determining the pathogenicity of small telomeric rearrangements may be more difficult. 3. It may be necessary to adjust the ratio of each mix in order to make the peak heights of all loci obtained consistent. For more product details, please visit Aladdin Scientific website.
PCR Primers Magnesium Chloride Solution dATP, dGTP, dCTP, and dTTP Mix Taq Polymerase 96-Well Plate PCR Thermal Cycler Agarose EB Solution Sampling Buffer Electrophoresis Buffer Long Ranger™ Gel Solution Urea
Horizontal Gel Electrophoresis Unit UV Transmission Reflectometer Filter Membranes GS-400 HD ROX ABI 377 Automated Sequencer
