Microarray (aCGH) Flashcards
Microarray
a molecular cytogenetic technique that allows precise and rapid detection of clinically relevant genomic imbalances, DNA copy number variations (CNVs), and mosaic abnormalities. It’s also called “Molecular Karyotyping” or “array Comparative Genomic Hybridization (aCGH)” by many cytogenecists. CMA can effectively diagnose birth defects caused by submicroscopic genomic imbalances in the neonatal period. It could confirm suspected chromosome abnormalities by karyotyping or FISH. CMA was about three times as likely to detect an abnormality than routine clinical chromosome analysis in a neonate referred with a clinical suspicion of a chromosomal syndrome. The technique is extremely useful when chromosome preparations are difficult or impossible. Since it measures the relative copy number of DNA sequences but not whether they have been translocated or rearranged from their normal position in the genome, balanced translocations, such as inversions, insertions, or low level mosaicism could not be detected. Greater resolution of this technique allows detection of genetic abnormalities before phenotype presents itself. Its application quickly expands into new areas such as cancer diagnosis, treatment options, and stem cells research with more targeted diagnostic arrays on their way.
G-banding
Genomic resolution: >4mb Whole genome approach:yes Detect balanced rearrangement:yes Tissue culture:yes Image interpretation:yes
FISH
Genomic resolution: 4-500 kb Whole genome approach:no Detect balanced rearrangement:yes Tissue culture:yes/no Image interpretation:yes
aCGH
Genomic resolution: 4-500 kb Whole genome approach:yes Detect balanced rearrangement:no Tissue culture:no Image interpretation:no
DNA sequencing
Genomic resolution: 1bp Whole genome approach:yes Detect balanced rearrangement:no Tissue culture:no Image interpretation:no
Types of array:
1.BAC array
An array of thousands of Bacterial Artificial Chromosome (BAC) clones representing regions of interest in the genome. Each of selected bacterial clones contains a BAC with a unique piece of human genomic DNA, at average ~ 150 Kb. The process includes growing large quantities of bacteria and isolating the BAC DNA, chemically modifying the BAC DNA, and printing the BAC DNA on glass slides. BAC array has lower resolution and lower probe specificity compared to oligo array. Cross-hybridization could cause false positive. Using dye-swap and excess amount of Cot-1 DNA could be costly. However, BAC array tends to work better with cancer samples than other types of arrays due to the mosaic nature of tumors.
Types of array:
2.Oligo array
An array of tens of thousands of oligonucleotide probes representing packed regions of interest in the genome, as well as backbone coverage at a lesser density for most other regions of the genome. Oligos are selected based on melting temperature (Tm), and specially designed for aCGH by trimming to 60 nucleotide bases (60 mers). Oligos are also searched for sequencing homology to the human genome to avoid cross-hybridization. Only unique Oligos are selected for inclusion in the high density (HD) arrays. Arrays were synthesized using ink-jet technology with phosporamide chemistry directly on glass slide. A custom clinical array with 44,000 oligos could equivalent to about 1400 BAC clones, with an average of 28 oligos /clone. Increased density of data points results in higher resolution which is equivalent to 6000 bands per haploid genome. Oligo array has greater dynamic range (signal to noise) due to use of strict selection criteria and removal of repetitive sequences. The process is simplified compared to BAC array therefore the overall cost is lowered. However, oligo array could not detect uniparental disomy (UPD) or copy neutron loss of heterozygosity (LOH).
Types of array:
3. SNP array
An array contains immobilized nucleic acid sequences for the detection of single site DNA variation (single nucleotide polymorphisms - SNP) within a population. More than 10 million SNPs have been identified in the human genome. SNP array has the highest theoretical resolution among three platforms and could detect disease-susceptible alleles. SNP array could detect UPD or copy neutron LOH which might be pathological. It could also be used to evaluate allelic imbalance among different types of cancer. The reference is publicly available from HapMap.
Clinical Applications
Clinical target aCGH in pre and postnatal diagnosis to detect unbalanced chromosomal abnormalities, cryptic unbalanced translocations, and the origin of supernumerary marker chromosomes.
Multiple congenital abnormalities.
Clinical suspicion of a chromosomal syndrome.
History of infertility.
Two or more unexplained miscarriages.
Unexplained mental retardation.
Maternal age - prenatal studies.
Family history of chromosome rearrangement.
Whole genome or tiling array in identifying CNVs in acquired genomic changes.
Molecular characterization of tumors and their subtypes.
Identification of novel chromosomal regions for therapeutic targets and new treatments.
Detect abnormalities not detected by karyotype (and vice versa)
Estimate the frequency of detecting CNV of uncertain significance
Advantages
Comparable to thousands of FISH experiments.
Offers whole-genome perspective at a much higher resolution than traditional Karyotyping.
Does not require culturing of cells.
Data can be generated by technicians with general lab skills.
Data can be processed & presented automatically: tables & graphs
Less subjective data interpretation.
Quick turn-around time (TAT) (~1.5 days).
Sensitive to low level mosaicism.
Limitations
Relatively expensive.
Not informative for balanced rearrangements and polyploidy.
The detection of copy number variations of uncertain significance is challenging to cytogeneticists.
Difficult to detect small subclones (constituting ~30% of sample or less).
Samples Used for aCGH
-Prenatal
CVS or Amniocentesis (direct analysis).
Cultured cells (from CVS or amniotic fluid).
Fetal blood.
-Postnatal
Peripheral blood.
Tissue biopsies.
Cultured cells.Procedure
Procedure
DNA from the patient sample such as blood, bone marrow, biopsy, or pregnancy is extracted. Commercially available DNA extraction kits and robotic DNA extraction machines are commonly used in diagnostic laboratories. Quality control is performed before amplification and labeling. Sample DNA is labeled with a red fluorescent dye (Cy5) and a reference DNA is labeled with green (Cy3). Sample needs to be cleaned up to remove excess dyes, random primers, and nucleotides. The two samples are co-hybridized to an array of genomic DNA spotted on a glass slide. Duration of hybridization could range from 8 to 48 hours based on platform used. Slide is washed in salt solutions and dried before scanning. Slide is scanned into image files using a scanner that could measure the intensity of the fluorescent signals for each spot on array. The areas on the slide that appear red indicate gain of extra chromosomal material in the test sample. Areas that appear in green indicate a loss in the sample. An output of scanning shows hundreds of spots with different ratio of the fluorescence intensities. Data is imported to software program designed to generate plot that reflects gain and loss for all regions included on the array. Pic on slide 11
How to Read Data
For each patient sample, two experiments are performed with a dye-swap for the test sample and gender-matched control. Data is subjected to normalization and the dye-reversed results are integrated to determine inferences for each clone. The log2 test/reference intensity ratio is used to objectively determine whether a clone deviates significantly from the baseline. Abnormal results are classified as gain or loss. A gain is defined when log2 ratio of a clone is greater than 1 whereas a loss is defined by log2 ratio < -1. Pic on slide 12
Example of Data
page 13
