Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Summary

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

Abstract

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient’s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Introduction

It has been known for many years that deletions or extra copies of chromosomal segments cause intellectual disability, dysmorphism and congentical malformations, and in some cases cause genetic syndromes¹. However, the only technology available until the mid-2000s for the genome-wide detection of these changes was G-banded chromosome analysis, which has a resolution of around 5-10Mb, depending on the region and nature of the imbalance, and which cannot detect abnormal chromosomes where material has been replaced by a region from a different chromosome with the same banding pattern. Ancillary cytogenetic techniques such as fluorescence in situ hybri....

Protocol

1. Labelling Reaction

1. Prior to use, pre-aliquot cyanine 3 and 5-labeled dUTPs, unlabeled nucleotides and random primers at the manufacturer-recommended concentrations into 96-well plates and store at -20 C ready for use. For the purposes of this protocol, aliquot 10.5 µl of the appropriate labeled dUTP and 10.5 µl unlabeled nucleotides and random primers to each well.
2. Thaw the ready-to-use 96-well plate of nucleotides and primers at 4 °C for about 1 hr, protected from light.
3. Once thawed, equilibrate this plate at RT for at least 30 min, protected from light.
4. Equilibrate DNA samples at 60 °C for at least 15 ....

Results

Each probe on a hybridized array is visualized as a mixture of red and green fluorochromes (see Figure 1). The ratio of red to green fluorescent signal for each probe is quantified by the scanner and the associated software plots these as log2 ratios according to their genomic position, and identifies regions falling outside preset boundaries. The resulting array traces allow interpretation of regions identified as genomically unbalanced. For instance, the trace from a child with Williams syndrome, a rec.......

Discussion

Array CGH will not detect balanced rearrangements or ploidy abnormalities such as triploidy. Furthermore, low level mosaic imbalances may not be detected. However, array CGH has a higher resolution for CNV detection than G-banded chromosome analysis which it has replaced in many cytogenetics laboratories. It is therefore the current gold standard for genome-wide CNV detection. It may be replaced by next-generation sequencing technologies in the future but currently, their cost and the technical complexities associated wi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CGH microarray 8 x 60K	Agilent	G4126
Array CGH wash buffers	Agilent	5188-5226
Array CGH hybridisation buffer	Agilent	5188-5380
Minelute purification kit	Qiagen	28006
Array CGH labelling kit	Enzo	ENZ-42672-0000