Experimental molecular detection of uniparental diploids

Uniparental diploid (UPD) cells can have a normal cytogenetic karyotype but an imbalance in parental contributions. The diagnosis of uniparental diploidy involves analyzing the genotype of DNA specimens from the patient and his/her parents, e.g., to assess the heritability of molecular polymorphisms. Microsatellite analysis, including PCR amplification of polymorphic short tandem repeat (STR) loci and polyacrylamide gel electrophoresis, is most commonly used for this purpose, distinguishing bands according to their size. Sometimes, uniparental diploids are only 'partial', i.e., include only one region of the chromosome. This requires careful selection of molecular markers to ensure that the uniparental diploid region is not missed. For some chromosomes, differences in methylation of homologous genes in both parents can also be used to screen for uniparental diploidy. The advantage of methylation methods is that parental DNA is not required and other imprinting defects can also be detected; the disadvantage is that uniparental diploidy cannot be distinguished from aberrant methylation due to other causes (e.g., the method itself).

Operation method

Molecular detection of uniparental diploids

Materials and Instruments

Sample DNA
Primer dNTP mixture Reaction buffer Taq DNA polymerase Acrylamide Methacrylamide TBE Ammonium persulfate
Thermocycler PCR tubes Electrophoresis unit

Move

I. Experimental Procedures for PCR Reactions

1. Prepare PCR reaction mixture: Determine the volume of the mixture other than DNA based on the number of DNA samples (add an extra portion to ensure that there is enough PCR reaction mixture) that will be amplified for each genetic marker and dispense 19.5uL of reaction mixture in each PCR reaction tube.

2. Add 0.5, of the DNA sample to each tube.

3. If you are not using a PCR instrument with a heated lid, add mineral oil to the top of each tube to prevent evaporation.

4. Place the tubes in the PCR instrument and heat at 95°C for 3 min to denature the DNA.

5. Denature at 95℃ for 40s, anneal at 55℃ for 40s, extend at 72℃ for 40s, and cycle 30-35 times.

6.72℃ final extension for 7 min.

7. The PCR product was mixed 1:1 with urea sample buffer.

1. Prepare gel solution: Add 25 g of urea, 7.5 mL of 40% acrylamide:PDA (19:1), and IOmL of 5XTBE to a 250 mL conical flask. Add distilled water to 50 mL. mix well to dissolve the urea (see Note 4).

2... Prepare the gel plate: Wash the plate - make sure it is free of dust. Use a siliconized glass plate. Depending on the manufacturer, clamp the glass plates together and seal the bottom with adhesive tape (this process varies depending on the equipment).

3. Add 500 uL of 10% ammonium persulfate to the gum mixture and filter through filter paper.

4. Remove 10mL of the gum solution and add it to a small beaker and add 10-15 buckets of TEMED. immediately pour the gum onto the plate. Within a few minutes the gum will polymerize and seal the bottom of the glass plate.

5. After sealing the glue, add 15ul of TEMED to the remaining glue solution and immediately pour the glue between the two plates. Carefully insert the glue comb and allow the glue to polymerize for 2 h to overnight.

6. Remove the seal from the bottom of the gel. Place the gel in the electrophoresis tank making sure that the electrodes are inserted in the correct direction. Fill the gel tank with 1XTBE. (approximately 1.5L, amount varies depending on the device). Remove the gel comb and flush the sample wells with IXTBE.

7. Preheat the gel for 60 min until the temperature is around 50 °C.

8. Rinse the sample wells again with the spiker before loading. Add 1?10/xL of product and sample dye to each well (sample volume varies depending on comb size and concentration of PCR product). Depending on the size of the product fragments, the run time can vary from 30 min to 3 h. The run time can be varied from 1 min to 2 h. The run time can be varied from 1 min to 3 h.

1. Remove the gel from the sequencing gel device using a 3 mm thick piece of blotting paper. The gel will stick to the filter paper and will not free until the gel is placed in a methanol/ice acetic acid solution.

2. Cut off the bottom of the gel so that it will fit in a heat-resistant baking dish without curling up on itself. Submerge the gum in enough solution to cover each step (approximately 300 mL). Avoid touching the gel with your hands as much as possible during the dyeing process. Lay the gel flat to avoid folding and lightly touch the corners of the gel with a gloved hand. Leave the gel in the dish during each step and pour off the old wash (you can gently pick up the gel with a piece of plastic sheet or a washed x-ray sheet with small holes in it).

3. Pour freshly prepared 10% methanol/10% glacial acetic acid into a heat-resistant dish and soak the gel for 15 minutes while gently shaking the heat-resistant dish to circulate the liquid.

4. Rinse the gel in 10% ethanol solution for 5 minutes.

5. Immerse the gel in 0.5% nitric acid solution for exactly 30s.

6. Rinse the rubber with water for 2 times.

7. Immerse the gel in 0.012mol/L silver nitrate solution for 15~30 min.

8. Rinse the gel quickly and thoroughly (rinse the gel twice with water, any silver nitrate that cannot be washed out will precipitate and produce spot deposits on the gel. The gel should not be immersed in water for too long because silver ions can also be washed out).

9. Immerse the gel in 300 mL of color developing liquid (when the liquid turns brown in color it should be replaced with new liquid).

10. Rinse again with water.

11. Immerse the gel in 10% glacial acetic acid for at least 2 min to terminate the reaction.

12. Rinse the gel with water.

13. Dry the glue between two pieces of cellophane with a glue dryer or place the glue on a 3 mm blotting paper and cover the top with wrapping paper.

1. DNA is usually extracted from 5~10mL of fresh whole blood. We usually choose salting out technique instead of phenol. Primary lysis products can be prepared if only a certain amount of sample needs to be extracted and no high quality DNA is required for other purposes. the DNA can also be extracted from other tissues, e.g., from simple oral rinses in quantities that can be used in multiple PCR reactions [24]. If only a very small amount of DNA is available, then pre-amplification of this DNA will be useful.

2. Primers can be synthesized by customization (if a large amount is needed).

3. Under standard amplification conditions, 3 cycles of DNA synthesis will be accomplished in 2 h. The primers can be custom synthesized (if large amounts are needed). There are several factors to consider when optimizing amplification conditions for specific primers:

(1) No or weak amplification: Try lowering the annealing temperature by a few degrees, doubling the primer concentration, gradually increasing the magnesium ion concentration or increasing the number of cycles by 3-5.

(2) Too much non-specific amplification: try to reduce the number of cycles, adjust the magnesium ion concentration, increase the annealing temperature by 2~5℃, and reduce the concentration of Taq enzyme.

(3) No or very weak primer dimer amplification products: Try reducing the primer concentration and increasing the steps mentioned above for non-amplification.

4. If silvering is not used, then normally use bis (methylenebisacrylamide, iV, V-methylenebisacrylamide) instead of PDA. PDA is a special cross-linking agent used to reduce the background of the silvering and at the same time to make the gel stronger, and this cross-linking agent is necessary to move the gel by hand during the silvering process. The crosslinker with bis can be used for silver dyeing, but due to the brittle nature of the gum, it can only be used for small gums.

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