Comparative Genomic Hybridization

Presented by-Vl-aM.ScDepartment of biotechnologyCentral Mizoram university

• Introduction•History•Basic methods •Application •Limitation

CONTENTS

• CGH is a molecular cytogenetic method for analyzing copy number variations (CNVs) relative to poidy level in the DNA for a test sample compared to a reference sample, without the need for culturing cells.

• The aim is to quickly and efficiently compare two genomic DNA samples arising from two sources which are mostly often closely related.

• This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resoIution of 5-10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized

Introduction of CGH

Introduction of CGH • In short, this involves the isolation of DNA from the two sources to be

compared, most commonly a test and reference source, independent labelling of each DNA sample with a different fluorophores (fluorescent molecules) of different colours (usually red and green), denaturation of the DNA so that it is single stranded, and the hybridization of the two resultant samples in a 1:1 ratio to a normal metaphase spread of chromosomes, to which the labelled DNA samples will bind at their locus of origin.

• Using a fluorescence microscope and computer software, the differentially coloured fluorescent signals are then compared along the length of each chromosome. A higher intensity of the test sample colour in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample colour indicates the loss of material in the test sample in that specific region. A neutral colour (yellow when the fluorophore labels are red and green) indicates no difference between the two samples in that location

History:

The first report of CGH analysis was by Kallioniemi and colleagues in 1992 at the University of california, San Franscisco, who utilised CGH in the analysis of solid tumors.

Soon after 1993, du Manoiret al. reported vertually same methodology. Applied CGH to genomic DNA from patients affected with either Downs syndrome or T-cell prolymphocytic leukemia as well as cells of a renal papillary carcinoma cell line.

(It was concluded that the fluorescence ratios obtained were accurate and that differences between genomic DNA from different cell types were detectable, and therefore that CGH was a highly useful cytogenetic analysis tool)

• Initially, the widespread use of CGH technology was difficult, as protocols were not uniform.

• However, in 1994 a review was published which described an easily understood protocol in detail and the image analysis software was made available commercially, which allowed CGH to be utilised all around the world.

• As new techniques such as microdissection and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) became available for the generation of DNA products, it was possible to apply the concept of CGH to smaller chromosomal abnormalities, and thus the resolution of CGH was improved.

Metaphase Slide Preparation

Isolation of DNA from Test Tissue and Reference

Tissue

DNA Labelling

Blocking

Hybridization

Fluorescence Visualisation and Imaging

Basic Methods

Metaphase Slide PreparationThe DNA on the slide is a reference sample, and is thus

obtained from a karyotypically normal man or woman

Isolation of DNA from Test Tissue and Reference TissueStandard phenol extraction is used to obtain DNA from

test or reference (karyotypically normal individual) tissue DNA Labelling

It is then important to check fragment lengths of both test and reference DNA by gel electrophoresis, as they should be within the range of 500kb-1500kb for optimum hybridization

BlockingUnlabelled Life Technologies Corporation's Cot-1 DNA® (placental

DNA enriched with repetitive sequences of length 50bp-100bp)is added to block normal repetitive DNA sequences, particularly atcentromeres and telomeres, as if these sequences are detected, they may reduce the fluorescence ratio and cause gains or losses to escape detection

Hybridization8-12µl of each of labelled test and labelled reference DNA are

mixed and 40 µg Cot-1 DNA® is added, then precipitated and subsequently dissolved in 6µl of hybridization mix, which contains 50% formamide to decrease DNA melting temperature and 10% dextran sulphate to increase the effective probe concentration in a saline sodium citrate (SSC) solution at a pH of 7.0

Fluorescence Visualisation and ImagingDedicated CGH software is commercially

available for the image processing step, and is required to subtract background noise, remove and segment materials not of chromosomal origin, normalize the fluorescence ratio, carry out interactive karyotyping and chromosome scaling to standard length

• The identification of chromosomal regions that are recurrently lost or gained in tumors, as well as for the diagnosis and prognosis of cancer. This approach can also be used to study chromosomal aberrations in fetal and neonatal genomes.

• Furthermore, conventional CGH can be used in detecting chromosomal abnormalities and have been shown to be efficient in diagnosing complex abnormalities associated with human genetic disorders.

Applications of CGH

• CGH in cancer research– CGH data from several studies of the same tumor type

show consistent patterns of non-random genetic aberrations.Some of these changes appear to be common to various kinds of malignant tumors, while others are more tumor specific.

• Chromosomal Aberrations– Cri du Chat (CdC) is a syndrome caused by a partial

deletion of the short arm of chromosome 5.Several studies have shown that conventional CGH is suitable to detect the deletion, as well as more complex chromosomal alterations.

• inability to detect structural chromosomal aberrations, without

copy number changes, such as mosaicism, balanced

chromosomal translocations, and inversions.

• CGH can also only detect gains and losses relative to the ploidy

level.

• The limited resolution of metaphase chromosomes, aberrations

smaller than 5–10 Mb cannot be detected using conventional CGH.

• The main disadvantage of array CGH is its inability to detect

aberrations that do not result in copy number changes and is

limited in its ability to detect mosaicism.

Limitations of CGH

• Another disadvantage is the lack of commercial availability of the arrays.

• However, array preparation still needs to be performed by the investigators themselves.

Inconsistencies in visualization and imaging software as well as interpretation parameters also make it difficult for replications and comparisons to be made by different laboratory teams

Thank You

Array CGH applications are mainly directed at detecting genomic

abnormalities in cancer. However, array CGH is also suitable for the

analysis of DNA copy number aberrations that cause human genetic

disorder. That is, array CGH is employed to uncover deletions,

amplifications, breakpoints and ploidy abnormalities. Earlier diagnosis is

of benefit to the patient as they may undergo appropriate treatments

and counseling to improve their prognosis

Genomic abnormalities in cancer

Submicroscopic aberrations

Prenatal Genetic Diagnosis

Array CGH