Micro-array based Comparative Genomic Hybridisation(aCGH)

Dr Yogesh D

Resident, Dept. of Anatomy

B J Medical College

Ahmedabad

Introduction

• Array Comparative Genomic Hybridisation (aCGH) is a molecular cytogenetic method for analysing copy number variations relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells.

Principle

• Fluorescent labelled DNA fragments from patient and control are applied to a microarray containing tens of thousands of probes fixed sequentially in triplets, and are allowed to competitively hybridise with the probes.

• Depending on the copy numbers of the DNA fragment, varying fluorescence is emitted that is scanned by an array scanner and an image is generated of the scan which is analysed by a software.

Method

• The sample collected from the patient is processed and the DNA is extracted.

• The entire genome is amplified by PCR.

• The amplified genome is fragmented, denatured and labelled with a fluorescent dye.

Method

• Similarly the control DNA fragments are labelled with a different coloured fluorescent dye.

• Equal quantities of labelled DNA fragments from both test and control are then applied on to the micro array.

• Microarrays contain tens of thousands of probes that are specifically engineered and range from 25 base pairs (oligonucleotides) to 200000 base pairs in length.

Method

• The DNA fragments from test and control are allowed to hybridise in PCR machine under incubation for 36 to 48 hours.

• Excess DNA fragments are washed off.

• The chip is scanned by the chip scanner and an image of the chip is generated.

• This image is then processed by a software and a graph is plotted.

Reporting Array CGH results

arr[hg19] 16p11.2(29,673,954-30,198,600 )x1

arr -- The analysis was by array-CGH

hg19 -- Human Genome build 19. This is the reference DNA sequence that the base pair numbers refer to. As more information about the human genome is found, new “builds” of the genome are made and the base pair numbers may be adjusted.

16p11.2 --- A change was found in band 16p11.2

Reporting Array CGH results

(29,673,954-30,198,600 ) -- The first base pair shown to be missing is number 29,673,954 counting from the left of the chromosome. The last base pair shown to be missing is 30,198,600

X1-- means there is one copy of these base pairs, not two – one on each chromosome 16 – as you would normally expect.

Interpreting Array CGH results

• Array CGH analysis of chromosome 6 for a human cancer cell line

Areas of application

• Prenatal Genetic Diagnosis.

• Cancer cytogenetics.

• Pre-implantation genetic diagnosis.

• Genetic screening of at risk parents.

• Mental retardation of unknown aetiology.

Advantages of aCGH

• Can be used to scan the entire genome for variations.

• Does not require actively multiplying cells.

• Varying probe sizes can be used depending on the requirement from as short as 25-80bp.

• The resolution offered by aCGH is very high.

• Submicroscopic chromosomal deletions and duplications can be easily detected.

Advantages of aCGH

• Can detect aneuploidies, deletions, duplications and/or amplifications simultaneously.

• One assay is equivalent to thousands of Fluorescent in situ hybridisation experiments.

• Particularly useful in the study of subtelomeric and pericentromericrearrangements.

Disadvantages

• Can miss balanced translocations.

• Can miss mosaicism.

• Does not identify tiny deletions or variations.

• Prohibitively expensive.

References

• Theisen, A. (2008) Microarray-based comparative genomic hybridization (aCGH). Nature Education 1(1):45

• Lobo, I. (2008) Chromosome abnormalities and cancer cytogenetics. Nature Education 1(1):68

• Kannan Thirumulu Ponnuraj (2011). Cytogenetic Techniques in Diagnosing Genetic Disorders, Advances in the Study of Genetic Disorders, Dr. Kenji Ikehara (Ed.), ISBN: 978-953-307-305-7, InTech,

• Chial, H. (2008) Cytogenetic methods and disease: Flow cytometry, CGH, and FISH. Nature Education 1(1):76

• Clancy, S. & Shaw, K. (2008) DNA deletion and duplication and the associated genetic disorders. Nature Education 1(1):23

• Eichler, E. E. (2008) Copy Number Variation and Human Disease. Nature Education 1(3):1