Comparative Genome Hybridization: A reliable replacement of conventional cytogenetic techniques... #genome #DNA #Biopharma #Medical
CGH is a cytogenetic assay which is used to determine chromosomal abnormalities such as additions and deletions. It is a combination of FISH and chromosomes analysis. It was first reported back in 1992 by Kallioniemi. Later on, the same methodology was used by Du Manior in 1993 with addition of using fluorophores for testing the technique.
Fluorescence ratio of labeled DNA molecules, one from normal cell and other from diseased cell, hybridized on metaphase chromosome slide is detected. The variation in the ratio of fluorescence tells about any deleted or extra chromosome.
1. METAPHASE SLIDE PREPARATION:
Heparinized peripheral lymphocytes are used to chromosomes. Phytohaemagglutinin is used to stimulate the cells. 1ml of heparinized blood is added to culture medium for 72 hours at 37°C in 5% CO2 condition. Colchicines are added to media to arrest the cell at metaphase as the chromosomes are most visible at this phase of cell division. Cells are then treated with KCl and fixed in 3:1 methanol/acetic acid. A drop of cell suspension is dropped on slide from 30 cm distance at 60-70% humidity conditions. Chromosomes are visualized under phase contrast microscope for any extra cytoplasmic content or impurity. Overlapping chromosomes should be removed as they are not recommended. If there is more cytoplasmic content in the slides it could create hindrance in showing results. To solve the problem, the slide is vertically washed with 3:1 methanol/acetic acid.
2. ISOLATION OF SAMPLE DNA AND REFERENCE DNA:
Next step is to isolate DNA one from normal cell and other from diseased cell of same individual. Normally the diseased cell may be a tumor cell. Simple phenol extraction can be used for this purpose. 0.5 to 1 microgram of DNA is sufficient for CGH.
3. LABELING OF DNA:
Next step is to label both sample and reference DNA with fluorophores such as cyanine 3 or cyanine 5. Multiple methods can be used to label DNA such as nick translation, direct labeling PCR, random primed labeling, cis-platinum labeling.
4. BLOCKING THE REPEATING SEQUENCES:
Sequences present at centromeres and telomeres must be blocked before hybridization because the length of such sequences can vary from individual to individual and may give increased or decreased values of fluorescence. For that purpose, we add Unlabeled Life Technologies Corporation's Cot-1 DNA®
Next is to hybridize the labeled DNA on the metaphase slide which was prepared initially. But before that, both the DNA and chromosomes must be denatured separately in order to hybridize them. Hybridization occurs under cover slips in humid conditions for two to four days. After hybridization, the slides are washed and stained with DAPI (4', 6-diamidino-2-phenylindole) in order to stain the chromosomes.
6. FLUORESCENCE VISUALIZATION AND IMAGING:
Fluorescence microscope is used for this purpose. Image is recorded with a camera of resolution 0.1 micrometer. Dedicated software is available commercially for processing of images. This relative copy number karyotyping tells about any deletions or additions.
LIMITATIONS OF CGH:
· CGH is unable to determine structural aberrations of chromosomes such as balanced chromosomal translocations, inversions and mosaicism.
· It is unable to determine the abnormalities smaller then 5-10 Mb
· It only detects gross level abnormalities
· CGH is used to determine aberrations in neonatal and fetal genomes
· It efficiently detects the genetic abnormalities relating to copy number
· Distinguishes between mild and metastatic tumors
· Used to analyze stillbirths having congenital abnormalities
· It analyzes stillbirths whose karyotypes cannot be obtained