Comparative genomic hybridization (CGH) experiments on microarray mid-split phase chromosomes

Summary

The unique advantage of chromosomal CGH is its whole-gene screening capability, which is significantly faster and less labor-intensive than low-throughput methods such as Southern analysis, PCR, and fluorescence in situ hybridization (FISH) that detect single-target DNA dose changes. Chromosomal CGH is currently a well-established molecular cytogenetic method, but its two shortcomings limit its use as a comprehensive screening tool.

Operation method

Microarray CGH Experiment

Materials and Instruments

High molecular mass genomic DNA EcoR I or Dpn II
SSC cDNA array dNTP mix dCTP yeast tRNA DNA polymerase I
Qiaquick PCR Purification Kit BioPrime Labeling Kit Micrcon 30 Filter Hybridization Oven Maxiprep DNA Extraction Kit

Move

I. cDNA array CGH

(1) Array preparation

Before hybridization with denaturing probes, the cDNA arrays were sealed with a sealing solution (covered with a cover slip) at 37°C for 1h

(2) Random primer labeling of genomic DNA

1. 2μg each of tumor and normal tissue genomic DNA was digested with Dnp II for 1~1.5 h. The digested products were purified (QiaquickPCR kit), vacuum-dried, and resuspended in 25μL of water.

2. Use the Bioprime labeling kit to label random primers basically according to the instructions, with slight modifications. denature the DNA at 100°C and 20 buckets of random primers (contained in the kit) for 5 min, quickly cool on ice, and add 2.5 μL of dNTP, 1.25 μL of dCTP, I μL of Cy3/Cy5-dCTP, and I μL of Klenow fragment (contained in the kit), and then add 2.5 μL of Klenow fragment (contained in the kit) at 37°C, and then add 2 μL of Klenow fragment (contained in the kit). contained in the kit), and incubate at 37°C for 90min.

3. Mix Cy3 and Cy5 labeling products and up-sample into a Micrcon30 filter. centrifuge at 2000g for 10min and inspect the sample container to determine the presence of labeling products (purple). Directly add 30 μL Cot-1 DNA, 100 μg yeast tRNA, 20 μg poly(dA-dT) and centrifuge at 5000 g for 20 min. To re-collect the samples, add 15 ml of hybridization buffer, invert the Micrcon30 filter into a new collection tube and centrifuge at 16,000 g for 1 min.

(3) Probe denaturation and hybridization

1. Denature the probe at 100℃ for 90s on a heated water bath or PCR instrument, cool the probe on ice and then revert at 37℃ for 0.5~1h.

2. Add the probe to the microarray, cover with a glass cover slip and seal the slice with rubber cement. Hybridize in a wet box moistened with hybridization buffer at 65°C for 16~20h.

(4) Elution

1. The cDNA microarray was washed in 2×SSC, 0.03% SDS at 65℃ for 5min, followed by elution with 1×SSC, 0.2×SSC each at room temperature for 5min.

2. Centrifuge at low speed (50g) for 5min to dry the specimen slice.

II. Array CGH

(1) Array preparation

1. Genomic clones (BAC, PAC, mucilage, etc.) are grown in the presence of appropriate antibiotics and genomic DNA is extracted in large quantities using a commercial kit, generally obtaining products of the order of 10 μg. The DNA is further purified by a standardized procedure using phenol/chloroform.

2. DNA size and quality were assessed on 1% agarose gel electrophoresis, and DNA was quantified by UV photometry.

3. The target DNA was fragmented by ultrasonic waves to obtain 1.5~15kb fragments, precipitated, resuspended to the appropriate concentration, and spotted onto glass carriers using capillary tubes with a diameter of 200~400μm.

4. Add 20μL of sealing solution to the array, cover the slide, and repeat at 37℃ in a wet box for 1h.

(2) Genomic DNA labeling by notch panning method for probe preparation

1. 2μg each of tumor and normal tissue genomic DNA was labeled by notch panning method, and the reaction mixtures were as follows:

A) Cy3 reactants (add water to a total volume of 100 μL)

(1) Tumor genomic DNA: 2 μg.

(2) 10×Cy3dNTP:10 μL.

(3)DNA polymerase:1μL.

(4) DNaseI.

B) Cy5 reactants (add water to a total volume of 100 uL).

(1) Normal human genomic DNA: 2 μg.

(2) 10×Cy5dNTP: 10 μL.

(3) DNA polymerase 1μL.

(4) DNaseI.

2. After the labeling reaction at 16°C for 1.5 h, the labeling reaction mixture was placed on ice.

3. The size of the labeled product was evaluated in 1% agarose gel electrophoresis, and the ideal fragment length for performing CGH experiments was in the range of 500~2000bp. If the fragments are too long, add appropriate amounts of DNA polymerase I and DNaseI to the reaction mixture and continue the reaction at 16°C.

4. Add 0.1 times the volume of 0.3 mol/LEDTA to the labeling reaction mixture to terminate the reaction.

5. Remove the unadulterated nucleotides from the labeling mixture by centrifugation on a Sephade×G50 column.

6. Mix the two labeled probes and add 50 μL of Cot-1 DNA, 0.1x volume of 3 mol/L sodium acetate, and 2x volume of ice-cold 100% ethanol to precipitate the probes. The precipitate was washed with 70% ethanol, air-dried, and then the probe was resuspended with 20 uL of hybridization buffer.

(3) Probe denaturation and hybridization

1. Denature the probe at 75°C for 5min, then revert at 37°C for 0.5~1h to adequately close the repeat fragment.

2. Add the probe to the array that has completed the pre-duplication, cover sheet and rubbermaid seal the sheet, and hybridize for 24h at 37℃ in a wet box moistened with hybridization buffer.

(4) Elution

1. The arrays were washed three times in 50% formamide/2×SSC, pH 7.0, at 55℃ for 10 min each time, and then washed in 0.1 mol/L sodium phosphate buffer containing 0.1% NP-40, pH 8, for 5~10 min at room temperature.

2. Remove the excess liquid, add DAPI/anti-quencher and cover the coverslip.

Caveat

1. There is controversy in establishing standardized terminology. While "probe" refers to the known nucleic acid sequence bound to the microarray, "target" refers to the unknown sequence of the sample. For the sake of consistency, this chapter follows the nomenclature currently used in all microarray CGH publications.

2. Before the advent of automated and practical batch extraction methods, many laboratories used bulk extraction kits to extract target DNA for microarray CGH preparation. this was a time-consuming and labor-intensive experiment that required repeated extractions of DNA when the target DNA ran out after multiple array spotting. if sufficient purified target DNA is available (at least a few hundred nanograms of template DNA should be available for either bulk or bulk preparation), DOP-PCR can be used to provide the target DNA. DOP-PCR can be used to provide an unlimited amount of target DNA.

3. It is critical to ensure that the microarray is moist throughout the hybridization process. Add hybridization buffer at any time to ensure that the hybridization cassettes are moist so that the probes or containment mixtures do not evaporate. If the microarray dries out, the results will not be reliable.

4. Listed here are optimized experimental protocols for cDNA microarrays up to 3500 samples within approximately (18X16) mm2 . When using higher density microarrays within a larger spot sampling area, the amount of DNA and the final hybridization volume must be proportionally higher.

5. In order to obtain high quality microarray CGH results, the size and purity of the unlabeled genomic DNA fragments are very important. The lower the purity of the DNA used for labeling, the higher the background and the weaker the hybridization signal of the microarray results obtained. The optimal experimental protocol for microarray CGH with genomic DNA extracted from fresh tissue is described here.

6. The choice of digestion with restriction endonucleases is important to improve labeling efficiency. It is worth noting that decreasing the average length of DNA fragments before labeling will improve labeling efficiency, while overdigestion yields probe fragments that are too small to be used for hybridization to cDNA targets. In our laboratory, very satisfactory and stable results were obtained using EcoRI digestion of human genomic DNA.

7. At the beginning of the establishment of the experimental method, a series of hybridization and elution temperature gradient experiments were performed to obtain the optimal hybridization intensity and specificity. It was found that 37°C hybridization and 55°C elution could detect high copy acquisition and amplification very sensitively, while increasing the elution temperature to 65°C would reduce the signal intensity of the microarray. Too low an elution temperature may produce nonspecific binding (excessive yellow signal). We recommend different labeling with sample DNA from different sources in preexperiments to get the best specificity for the experimental technique.

8. To date, there is still no standardized method for array preparation. Published papers with target DNA concentrations of 400 ~ 10 μg/mL were manually spotted on polylysine-treated slides, or Zyg/yL target DNA spotted on aminopropyltrimethoxysilane-treated slides [12]. Note that with the advent of automated programs for machine spotters, both the DNA concentration and slide treatment methods may change accordingly.

9.The methods mentioned here assume that a (22X20)mm2glass cover slip range to construct the maximum amount of small gridded spotting area. In addition, it is assumed that the target DNA has been denatured during array preparation. Otherwise, the microarrays must be denatured in 70% formamide/4×SSC for 2 min before hybridization with the probe10. The final length of the probe is dependent on the concentration of DNAzyme I. The optimal length of probes for CGH experiments is in the range of 500~2000bp. The enzyme is stored at a concentration of 1×10-4U/μL, and the final concentration of 5×10-5U/μL is freshly prepared with DNAzyme I diluent prior to the experiment. However, the enzyme concentration was adjusted accordingly as the length of the desired DNA fragment required was changed.

11. 0.05 ~ 0.1 times the volume of each labeling mixture was subjected to gel electrophoresis for DNA staining (e.g., ethidium bromide). Agarose gel electrophoresis is recommended for evaluating the markers since it can contribute to array CGH problem solving.

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