BioPrime Array CGH Genomic Labeling System - For generating fluorescently labeled genomic DNA to use in microarray screening

Materials Needed

The following materials are supplied by the user:

• 250 ng–1 μg genomic DNA (for BAC arrays) or 4 μg genomic DNA (for cDNA arrays); amount is arraydependent
• Microcentrifuge
• Vortex Mixer
• Aerosol-resistant pipette tips
• Amber 1.5-ml microcentrifuge tubes
• Incubators or water baths set at 95ºC and 37ºC
• Ice
• CyDye™ fluorescent dCTP or dUTP, or fluorescent dCTP/dUTP from another manufacturer
• 100% isopropanol and 100% ethanol

Description of the Kit

Purpose of the Kit

Comparative genomic hybridization (CGH) is a microarraybased method for analyzing the whole genome to detect variations in gene copy number between samples (Pollack et al, 1999, 2002). In CGH, two genomic DNA sample  are labeled with different fluorophores and hybridized to a microarray. The ratio of the fluorescent intensities of the fluorophores is measured for each gene on the array. This ratio provides a relative measure of the difference in gene copy number between the samples. The BioPrime® Array CGH Genomic Labeling System uses random primers and a mutant form of the Klenow fragment of DNA polymerase I (Exo– Klenow) to differentially label genomic DNA samples with fluorescently labeled nucleotides for analysis using CGH. Probes generated with thi  system can differentially detect differences in gene copy number from as little as 250 ng of genomic DNA. The kit is compatible with fluorescent nucleotides from a variety of manufacturers, including Cy3™- and Cy5™-labeled dNTPs and Alexa Fluor®-labeled dNTPs.

How to Use

Using the kit, you anneal your genomic DNA with random octamers. The primers are extended in a polymerization reaction using a high concentration of Exo– Klenow and fluorescently labeled nucleotides, resulting in high nucleotide incorporation and at least a 7–10-fold amplification of starting material. You then purify the labeled samples using spin columns to remove unincorporated nucleotides and proceed to microarray hybridization.

Advantages of the System

• Exo– Klenow polymerase lacks both 5’-3’ and 3’-5’ exonuclease activity, producing higher yields of labeled sample than standard Klenow for increased sensitivity.
• Exo– Klenow polymerase incorporates fluorescently modified nucleotides more effectively than standard Klenow, enabling you to obtain stronger hybridization intensities and greater reproducibility of results.
• Simplified random priming protocol takes less than three hours.

Workflow Overview

• Add each genomic DNA sample to be compared to a separate tube.
• Add random primers to each sample.
• Heat each mixture briefly to denature the DNA, then cool to anneal the primers.
• Add the appropriate 10X Nucleotide Mix, the appropriate dye, and Exo– Klenow to each tube.
• Incubate at 37ºC for 2 hours to amplify and label the DNA.
• Add Stop Buffer to stop each reaction.
• Add Binding Buffer B2 to each labeled sample, then spin, wash, and elute using the PureLink™ spin columns.
• Proceed to hybridization

Dye Compatibility

This kit has been developed using Cy3™- and Cy5™-labeled nucleotides from Amersham Biosciences. Fluorescently labeled nucleotides from other manufacturers (e.g., Alexa Fluor®-labeled dNTPs from Molecular Probes) are also compatible with this system.

Control DNA

Control DNA (salmon sperm DNA) is included in the kit to help you determine the efficiency of the labeling procedure. We recommend that you perform the complete labeling procedure using the Control DNA if you are a first-time user of the system. Equations for calculating the efficiency of the labeling procedure using the Control DNA are provided later on this page.

Product Qualification

The Certificate of Analysis provides detailed product qualification information for each production lot. Certificates of Analysis are available on our website. Go to the support page and search for the Certificate of Analysis by product lot number, which is printed on the box.

• Use amber microcentrifuge tubes as specified.
• Be careful to minimize exposure of the fluorescent nucleotides and labeled DNA to light.

• 250 ng–1 μg genomic DNA (for BAC arrays) or 4 μg genomic DNA (for cDNA arrays); amount is arraydependent
• Microcentrifuge
• Amber 1.5-ml microcentrifuge tubes
• Incubators or water baths set at 95ºC and 37ºC
• Ice
• CyDye™ fluorescent dCTP or dUTP, or fluorescent dCTP/dUTP from another manufacturer

1. Add each genomic DNA sample to be compared to a new amber 1.5-ml microcentrifuge tube and suspend in sterile distilled water to a final volume of 21 μl. Control reactions: Use 1 microgram (μg) of the Control DNA included in the kit (supplied at 10 μg/μl) per reaction.
   
   
2. Add 20 μl of 2.5X Random Primers Solution to each sample.
   
   
3. Incubate at 95ºC in an incubator or water bath for 5 minutes, and then immediately cool on ice for 5 minutes.
   
   
4. On ice, add the following to each tube to differentially label each sample:
   
   

   	DNA 1	DNA 2
   10X dCTP or dUTP Nucleotide Mix	5 μl	5 μl
   Cy3™-dCTP or -dUTP	3 μl	-
   Cy5™-dCTP or -dUTP	--	3 μl
   Exo– Klenow Fragment	1 μl	1 μl

   
   
   
   
5. Mix gently and perform a quick spin down (5 seconds).
   
   
6. Incubate at 37ºC for 2 hours.
   
   
7. Add 5 μl of Stop Buffer to each tube and place on ice. The reaction can be stored at –20ºC overnight, if necessary.

1. Add 200 μl of  inding Buffer B2 (prepared with isopropanol as described above) to each tube from Step 7, above, and vortex to mix.
   
   
2. Load each sample onto a PureLink™ Spin Column, preinserted in a collection tube.
   
   
3. Centrifuge at 10,000 × g for 1 minute. Discard the flowthrough and place the column back in the collection tube.
   
   
4. Add 650 μl of Wash Buffer W1 (prepared with ethanol as described on page 6) to the column.
   
   
5. Centrifuge at 10,000 × g for 1 minute. Discard the flowthrough and place the column back in the collection tube.
   
   
6. Spin at maximum speed for an additional 2–3 minutes to remove any residual wash buffer. Discard the flow through.
   
   
7. Place the Spin Column in a new, sterile Amber Recovery Tube (supplied in the kit).
   
   
8. Add 55 μl of Elution Buffer E1 to the center of the column and incubate at room temperature for 1 minute.
   
   
9. Centrifuge at maximum speed (~20,000 × g) for 2 minutes. The flow-through contains the purified labeled DNA probes. (Discard the column after use.)  To determine the efficiency of the labeling reaction, proceed below.

Calculating the Results

To calculate the amount of labeled DNA using a UV/visible spectrophotometer:

1. Transfer an appropriate volume of purified, labeled DNA from step 9, above, to a clean cuvette. Use an appropriate volume for your spectrophotometer. Blank the spectrophotometer using 10 mM Tris-HCl, pH 8.5.
   
   Important: The labeled DNA must be purified before scanning, as any unincorporated labeled nucleotides will interfere with the detection of labeled DNA.
   
   
2. Measure the absorbance of the sample at A₂₆₀, A₃₂₀, A₅₅₀, A₆₅₀, and A₇₅₀. Wash each cuvette thoroughly between samples.

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2. Cai, W.W., Mao, J.H., Chow, C.W., Damani, S., Balmain, A., and Bradley, A. (2002) Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays. Nature Biotechnology 20. 393–396.
   
   
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