Methods

DNA Extraction

High-molecular-weight whole genomic DNA (>4 kb) was obtained for reference DNA from healthy female and male donors and also for test DNA from samples. The normal reference DNA was prepared from peripheral lymphocytes, and the test DNA was from tissue samples. DNA was extracted after overnight proteinase K digestion followed by the phenol chloroform isoamyl method and alcohol precipitation.

The concentration of reference and test DNA were measured with a fluorometer.

Preparation of Metaphase Spreads

The quality of the normal metaphase spreads, to which reference and test DNA were hybridized, strongly influence the reliability and sensitivity of CGH analyses. Metaphase spreads were prepared from synchronized cultures of peripheral blood cells from a healthy male donor (Fig. 36-3). T lymphocytes in RPMI 1640 medium were stimulated with phytohemagglutinin and cultured for 72 hours. The cells were then synchronized by treatment with 10~7 M methotrexate (MTX) for 15 hours to inhibit DNA replication, followed by 10"5 M thymidine for 5 hours to release the cells synchronously from the MTX-induced block. Colcemid (1 pg/mL) was added during the final 30 minutes of thymidine release. Lymphocytes were fixed in a 3:1 solution of methanol and acetic acid and dropped on precleaned microscope slides. The slides were air-dried using a Thermotron environmental chamber.

Comparative Genomic Hybridization

CGH was performed according to the protocol described by Kallioniemi and associates,6 with slight modifications using fluorochromes conjugated to dUTP for standard nick translation (see Fig. 36-2).8 Test and reference DNA were labeled using the nick translation reaction with fluorescein-12 (FITC)-dUTP and Alexa Fluor 568-5-dUTP, respectively. The size of DNA fragments was adjusted from 500 to 1500 bp for hybridization, depending on the amount of DNA polymerases and incubation time. Approximately 200 ng each of FITC-labeled test and Alexa-568-labeled reference DNA samples were hybridized to the normal metaphase spreads. Twenty micrograms of unlabeled Cot-1 DNA was used to block the binding of repeated DNA sequences. The DNA was denatured for 5 minutes at 73°C in hybridization solution (50% formamide, 10% dextran ras

Normal thyroid follicular cell

Follicular adenoma p16

ret, trk

Occult papillary carcinoma

Follicular carcinoma p16

Papillary carcinoma p53 I

Anaplastic carcinoma

FIGURE 36-1. Carcinogenesis in thyroid tumors.

G2 Phase 3-4 hrs.

FIGURE 36-3. Schema of cell cycle.

G2 Phase 3-4 hrs.

M Phase

L t : '

1 hr.

"* M- *

Ö ' T<'

\

y i ,

FIGURE 36-3. Schema of cell cycle.

sulfate, and 2X SSC, pH 7.0). Metaphase slides were also denatured in a denaturing solution (70% formamide, 2X SSC, pH 7.0) for 3 to 5 minutes at 73°C and dehydrated with ethanol. Hybridization was performed in a chamber at 37°C for 2 or 3 days. Posthybridization slides were washed three times in washing solutions (50% formamide, 2X SSC, pH 7.0), once in 2X SSC at 45°C, once in 2X SSC at room temperature, twice in a PN buffer (0.1 M Na2HP04, 0.1 M NaH2P04, 0.1% NP-40, pH 8.0), and once in distilled water at room temperature. The slides were counterstained with 10 pL of 0.4 pM 4',6-diamidino-2-phenylindole (DAPI) in an antifade solution.

Digital Image Acquisition and Analysis

The three-color images—blue (DAPI), green (FITC), and red (Alexa-568)—with appropriate light source and filters were acquired using several different image acquisition systems (Figs. 36-4 and 36-5). At least 10 images of metaphase spreads were used for each hybridization. These three-color images were analyzed to determine the ratio of green and red fluorescence intensity along each chromosome. Image analysis typically involved normalizing the intensity of green and red images, chromosome segmentation, background subtraction, medial axis calculation, integration of fluorescence intensity in bands perpendicular to the medial axis across each chromosome, and calculation of green-to-red ratios along each medial axis. The green-to-red ratio indicated the relative DNA sequence copy number at each point in the test genome. At least six metaphase spreads were analyzed per hybridization and the results were averaged. The regions with a green-to-red ratio of more than 1.20 were interpreted as gains and those with a ratio less than 0.80 as losses. However, the results were dependent on the cutoff values. Cot-1 DNA inhibited binding of the labeled DNA to the centromeric and heterochromatic regions, so that the centromeric areas of chromosome 1,9, 16, and Y and the

Nick translation with green fluorescence (FITC)

Tumor DNA

Cot-1 DNA

Nick translation with red fluorescence (Alexa-568)

Normal DNA

FIGURE 36-2. Technique of comparative Hybridization genomic hybridization. Equal amounts of the fluorochrome-labeled test and reference DNA were hybridized to normal metaphase spreads with unlabeled Cot-1 DNA to block the binding of repeated DNA sequences.

Normal metaphase chromosomes

Normal metaphase chromosomes

Normal metamorphosis chromosomes

Normal

Gain

Loss

U1 c

Normal

Gain

FIGURE 36-4. Schema in digital image acquisition and analysis.

Mixed red/green fluorochromes

(yellow-orange)

Probe labeled with green fluorescence

Probe labeled with red fluorescence p arm of acrocentric chromosomes (chromosomes 13-15, 21, and 22) could not be analyzed in this study. A positive control with known chromosomal abnormalities and a negative control using normal human male and female DNA were used in each hybridization as controls to verify the reliability of this method.

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