Introduction

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.

Ordering Information

Catalog # Name Size List Price (USD) Qty
18095011 BioPrime® Array CGH Genomic Labeling System 30 reactions 426.00
18095012 BioPrime® Array CGH Genomic Labeling Module 30 reactions 386.00

Methods - Preparing the DNA

Starting Material

This kit is optimized for use with 250 ng–1 μg genomic DNA (for BAC arrays) or 4 μg genomic DNA (for cDNA arrays) as starting material. In general, use the amount of starting material recommended by your array manufacturer.

Isolating Genomic DNA

Isolate genomic DNA using your method of choice. The PureLink™ Genomic DNA Purification Kit (K1810-01) is a complete kit for the isolation of genomic DNA. See page 21 for ordering information. A wide range of ChargeSwitch® Genomic DNA purification kits is also available from Invitrogen. DNA Digestion/Sonication Genomic DNA may be either intact or treated by enzymatic digestion or sonication, depending on the requirement  of your array manufacturer. Note that DNA that has been fragmented by enzymatic digestion or sonication generally yields better results in array CGH.

General Handling of DNA

When handling DNA, use sterile conditions to ensure that no DNases are introduced. All equipment that comes into contact with DNA should be sterile, including pipette tips, microcentrifuge tubes, snap-cap polypropylene tubes, and pipettes. Be sure pipettor barrels are clean and treated with ethanol.

Checking DNA Quantity and Quality

Genomic DNA may be run on an agarose gel to check for quantity and quality. Bufferless E-Gel® Pre-cast Agarose Gels are available from Invitrogen for fast and easy electrophoresis. See page 21 for ordering information. Storing DNA After isolating the DNA, we recommend that you proceed directly to Labeling on the next page. Otherwise, store the isolated genomic DNA at +4°C.
TOP

Labeling

Introduction

This section provides a protocol for labeling genomic DNA with fluorescently labeled nucleotides. The protocol uses CyDye™ fluorescent nucleotides, and is compatible with fluorescent nucleotides from other manufacturers.

Dye Information

This system has been developed using the following CyDye™ fluorescent nucleotides:

Cy3™-dCTP (Amersham Biosciences, #PA53021)
Cy3™-dUTP (Amersham Biosciences, #PA53022)
Cy5™-dCTP (Amersham Biosciences, #PA55021)
Cy5™-dUTP (Amersham Biosciences, #PA55022)

It is also compatible with fluorescent nucleotides from other manufacturers.

Fluorescent nucleotides are sensitive to photobleaching. When preparing the reaction:

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

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
  • 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

Preparing the Control DNA

Use 1 μg of Control DNA per reaction. The Control DNA is provided at a concentration of 10 μg/μl; to avoid pipetting 0.1 μl, we recommend diluting 1 μl of the control 1:10 in sterile distilled water prior to use.

Incubation Methods

The incubation steps may be performed in a heat block, air incubator, or thermocycler with a heated lid. Incubate the reaction protected from light.

Nucleotide Mixes

Select the type of nucleotide mix based on the type of dyelabeled nucleotides you are using. Use the 10X dCTP Nucleotide Mix with labeled dCTP and 10X dUTP Nucleotide Mix with labeled dUTP.

Labeling Procedure

Follow the steps below to incorporate the fluorescent nucleotides in the genomic DNA.

  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 Mix5 μl5 μl
    Cy3™-dCTP or -dUTP3 μl-
    Cy5™-dCTP or -dUTP--3 μl
    Exo– Klenow Fragment1 μl1 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.

Proceed to Purification below
TOP

Purification

BioPrime® Purification Module

Catalog no. 18095-011 includes a purification module with PureLink™ spin columns and buffers. Follow the procedure in this section to purify your labeled DNA using this module.

Other Purification Methods

Catalog no. 18095-012 does not include a purification module. Use your preferred method of purification, and then proceed to page 16. When assessing the labeling efficiency using a spectrophotometer, be sure to blank the spectrophotometer using the elution buffer from your purification system. Note: The PureLink™ PCR Purification System can be ordered separately if you are using catalog no. 18095-012.

Purification Procedure


Follow the steps below using the Purification Module from catalog no. 18095-011 to purify the labeled DNA probes.

  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.
TOP

Assessing the Efficiency of the Labeling

CyDye™ Wavelengths

The following table shows the absorbance and baseline wavelengths for Cy3™ and Cy5™ dyes:

Dye Absorbance Wavelength Baseline Wavelength
Cy3™550 nm650 nm
Cy5™650 nm750 nm


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.


Yield:1
DNA (μg) = (A260–A320) × 50 μg/ml × volume in ml
Dye Incorporation:2
Cy3™ (pmole) = (A550–A650)/0.15 × volume in μl
Cy5™ (pmole) = (A650–A750)/0.25 × volume in μl
Degree of Labeling:
Base/dye for Cy3™ = (A260 × 150,000 (cm–1 M–1))/(A550 × 6,600)
Base/dye for Cy5™ = (A260 × 250,000 (cm–1 M–1))/(A650 × 6,600)
Notes:
1Subtracting A320 from A260 corrects for any silica particles that may leak from the purification columns and artificially increase the yield calculations.
2Subtracting A650 from A550 and A750 from A650 corrects for any fluorescent background that might artificially increase the measure of dye incorporation.

Expected Results

Using the calculations on the previous page, you should expect the following: Yield: The expected amount of labeled DNA should be ≥ 2.8 μg. If it is < 2.8 μg, see Troubleshooting.

Dye incorporation: The expected level of dye incorporation should be ≥100 pmoles. If it is < 100 pmoles, see Troubleshooting.

Base/dye ratio: The base-to-dye ratio should be 40–80 for both Cy3™ and Cy5™.

Dye Incorporation versus Signal Intensity

Signal intensity and signal/background on microarrays do not correlate directly with dye incorporation or degree of
labeling when comparing different fluorescent dyes. Labeling with the BioPrime® Array CGH Genomic Labeling System yields microarray signal intensities and signal/background ratios greater than or equal to DNA labeled with other dye-labeled nucleotides, even with lower dye incorporation and/or degree of labeling.
TOP

Troubleshooting

Problem Cause Solution
Yield of labeled DNA from the control reaction is lowDNA has been lost in the purification step after labelingMake sure that isopropanol has been added to the Binding Buffer and ethanol has been added to the Wash Buffer. Measure the amount of labeled DNA in the control reaction before and after purification. Repeat the labeling and purification procedures, following all steps without modifications.
 Starting amount of DNA is too lowIncrease the amount of starting DNA.
Cannot detect labeled probesDNA has been lost in the purification step after labelingMake sure that isopropanol has been added to the Binding Buffer and ethanol has been added to the Wash Buffer. Measure the amount of labeled DNA in the control reaction before and after purification. Repeat the labeling and purification procedures, following all steps without modifications.
Amount of incorporated labeled nucleotides is low or fluorescence is lowStarting amount of DNA is too lowIncrease the amount of starting DNA
 Reaction tubes have been exposed to lightAvoid direct exposure of the reaction tubes to light. Repeat the labeling procedure.
 Fluorescent nucleotides have been exposed to lightRepeat the labeling reaction, being careful to avoid direct exposure to light.
 Inefficient labeling due to improper purificationFollow all the purification steps as described in the procedures
TOP

References

  1. Pollack, J.R., Perou, C.M., Alizadeh, A.A., Eisen, M.B., Pergamenschikov, A., Williams, C.F., Jeffrey, S.S., Botstein, D., and Brown, P.O. (1999) Genomewide analysis of DNA copy-number changes using cDNA microarrays. Nature Genetics 23. 41–46.

  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.

  3. Pollack, J.R., Sorlie, T., Perou, C.M., Rees, C.A., Jeffrey, S.S., Lonning, P.E., Tibshirani, R., Botstein, D., Borresen-Dale, A.L., and Brown, P.O. (2002) Microarray analysis reveals a major direct role of DNA copy number

  4. alteration in the transcriptional program of human breast tumors. Proc. Natl. Acad. Sci. USA. 99. 12963–12968.

  5. Snijders, A.M., Nowak, N., Segraves, R., Blackwood, S., Brown, N., Conroy, J., Hamilton, G., Hindle, A.K., Huey, B., Kimura, K., Law, S., Myambo, K., Palmer, J., Ylstra, B., Yue, J.P., Gray, J.W., Jain, A.N., Pinkel, D., and Albertson, D.G. (2002) Assembly of microarrays for genome-wide measurement of DNA copy number. Nature Genetics 29. 263–264

  6. Hodgson, G., Hager, J.H., Volik, S., Hariono, S., Wernick, M., Moore, D., Nowak, N., Albertson, D.G., Pinkel, D., Collins, C., Hanahan, D., and Gray, J.W. (2001) Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas. Nature Genetics 29. 459-464

  7. Beheshti, B., Braude, I., Marrano, P., Thorner, P., Zielenska, M., and Squire, J.A. (2003) Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization. Neoplasia 5. 53–62

  8. Pinkel, D., Segraves, R., Sudar, D., Clark, S., Poole, I., Kowbel, D., Collins, C., Kuo, W.L., Chen, C., Zhai, Y., Dairkee, S.H., Ljung, B.M., Gray, J.W, and Albertson, D.G. (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nature Genetics 20. 207–211
TOP
MAN0000434         2-Nov-2009