The document discusses array comparative genomic hybridization (aCGH), a method for analyzing DNA copy number variations without cell culturing. It details the principles, methods of sample processing, and application areas, such as prenatal genetic diagnosis and cancer cytogenetics. Advantages include high-resolution detection of chromosomal abnormalities, while disadvantages encompass potential missed translocations and high costs.

1. Micro-array based Comparative
Genomic Hybridisation(aCGH)
Dr Yogesh D
Resident, Dept. of Anatomy
B J Medical College
Ahmedabad

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

8. 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

9. 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.

10. Interpreting Array CGH results
• Array CGH analysis of chromosome 6 for a human
cancer cell line

11. Areas of application
• Prenatal Genetic Diagnosis.
• Cancer cytogenetics.
• Pre-implantation genetic diagnosis.
• Genetic screening of at risk parents.
• Mental retardation of unknown aetiology.

12. 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.

13. 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 pericentromeric
rearrangements.

14. Disadvantages
• Can miss balanced translocations.
• Can miss mosaicism.
• Does not identify tiny deletions or variations.
• Prohibitively expensive.

15. 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