CMC currently offers four Affymetrix GeneChip-based genomic testing. Each link below provides basic information about the test, including background, rational and method. Given the rapid development of this emerging field, we plan to expand our services to include those listed under upcoming services.
Current Services
Differential Gene Expression Assay Gene expression is tightly regulated. Disruptions in gene expression are responsible for many diseases. The global gene expression profiling will aid in identifying disease-cause or associated genes/signature.
Gene alternative splicing assay Approximately 30-60% of genes undergo alternative splicing. Abnormal splicing can cause severe diseases. This assay will identify gene splicing variants and their potential disease association.
Copy Number Variation Assay-array CGH Copy number variation in the human genome is widespread. This assay is designed to identify the pathogenic copy number variants.
High-throughput Genotyping Assay Single nucleotide polymorphic markers have revolutionized the high-density genotyping. This assay can be used in the genome-wide association study and linkage analysis to identify markers/genes linked to the pathogenesis of a wide range of diseases.
Future Services
- AmpliChip CYP450 Genotyping Test
- Tissue of Tumor Origin Test
- AmpliChip p53 Test
- Amplichip Leukemia assay
- Amplichip Lymphoma assay
Differential Gene Expression Assay "back to top"
Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. This mechanism acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes as necessary. The harmonious expression profile is a critical component of maintaining human health. Disruptions or changes in gene expression are responsible for many diseases. Thus, examining global gene expression profile under particular physiological or pathological condition will provide insight into gene function, mechanisms of disease and aid in identifying disease related signatures or profiles that can be applied to diagnosis, drug target selection and monitoring response to therapy.
The Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array is the most comprehensive tool to study global gene expression. It consists of 1,300,000 unique oligonucleotide features covering over 47,000 transcripts and variants, and representing approximately 39,000 of the best characterized human genes. This array can determine the expression level of all well annotated as well as less annotated human genes in a single assay,
The global gene expression assay includes five key steps:
- The Total RNA (1 µg to 15 µg) is first reverse transcribed using a T7-Oligo(dT) Promoter Primer in the first-strand cDNA synthesis reaction.
- Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA is purified and used as a template in the subsequent in vitro transcription (IVT) reaction.
- The IVT reaction is carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling.
- The biotinylated cRNA targets are then cleaned up, fragmented, and hybridized to GeneChip expression arrays.
- The hybridized array is stained with a streptavidin phycoerythrin conjugate and scanned by the GeneChip® Scanner 3000DX V.2. The amount of light emitted at 570 nm is proportional to the bound target at each location on the gene array.
Initial levels of gene expression are reviewed by using Affymetrix software. Subsequent advanced data analysis will be performed to identify statistically, differentially expressed genes/signature potentially responsible for or associated with the physiological or pathological conditions.
Gene alternative splicing assay "back to top"
Recent studies have estimated that approximately 30-60% of genes undergo
alternative splicing , which represents an important regulatory mechanism. Abnormal splicing can produce functionally distinct proteins and sometimes cause severe diseases. Scientists are trying to determine the cause of aberrant splicing and to understand its disease association. Recent literature has demonstrated a clear link between a mis-spliced gene and a disease state including a vast array of neurological and autoimmune diseases.
Affymetrix GeneChip® Human Exon 1.0 ST Array enables genome-wide analysis of alternative splicing. It is a single array with over 5 million unique 25-mer oligonucleotide features constituting 1.4 million probe sets with approximately four probes per exon and roughly 40 probes per gene. The array covers over 1 million exon clusters (1 or more overlapping exons) and over 150,000 transcript clusters (groups of putative alternatively spliced transcripts from the same gene). This exon-level analysis on a whole-genome scale opens the door to detecting specific alterations in exon usage that have been shown to play a central role in disease mechanism and etiology.
Gene alternative splicing assay consists of the following eight key steps:
- rRNA reduction using RiboMinus reagents to minimize the background and increase the array detection sensitivity and specificity.
- Followed by the double-stranded cDNA synthesis with random hexamers tagged with a T7 promoter sequence.
- The double-stranded cDNA is amplified by T7 RNA polymerase producing antisense cRNA.
- Random hexamers are used to prime reverse transcription of the cRNA to produce single-stranded DNA that contains dUTP in the sense orientation.
- Uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1)-mediated single-strand DNA fragmentation.
- The fragmented DNA is labeled by terminal deoxynucleotidyl transferase (TdT) with the Affymetrix® proprietary DNA Labeling Reagent.
- The biotinylated DNA targets are hybridized to Affymetrix GeneChip® Human Exon 1.0 ST Array.
- The hybridized array is stained with streptavidin phycoerythrin conjugate and scanned by the GeneChip® Scanner 3000DX V.2. The amount of light emitted at 570 nm is proportional to the bound target at each location on the gene array.
Initial levels of exon expression are analyzed by using Affymetrix software. Partek Genomics Suite will be subsequently used to identify splicing variants potentially responsible for or associated with the physiological or pathological conditions.
Copy Number Variation Assay "back to top"
A copy number variant is operationally defined as a DNA segment, longer than 1 kb, with a variable copy number compared with a reference genome. The copy number variation in the human genome is widespread. Many of the copy number variants (CNVs) are pathogenic.
The GeneChip® Human Mapping 500K Arrays provide a powerful tool for detecting CNVs. It is comprised of two arrays, each capable of genotyping on average 250,000 SNPs (approximately 262,000 for Nsp arrays and 238,000 for Sty arrays). The SNPs on the Arrays were chosen for even distribution across the genome with a mean marker distance of 5.6 kb, providing researchers with more power and confidence to detect changes in chromosomal copy number and more resolution to define aberration boundaries. Recently, Affymetrix® has released Genome-Wide Human SNP Array 6.0, which features more than 1.8 million markers for genetic variation, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes for the detection of copy number variation. The SNP Array 6.0 enables high-performance, high-powered and low-cost genotyping, thus particularly useful for analysis of diseases or genetic characteristics that arise from gene duplications, deletions, or other chromosomal abnormalities including mental retardation, renal disorders, diabetes, and autism. Data also are of potential value in diagnosis and management of cancers such as melanoma, where a correlation has been observed between disease grade and markers.
The assay involves the following 7 key steps for 500k arrays:
- 250 ng of genomic DNA is initially digested with the restriction enzymes Nsp I or Sty I.
- Then ligated to a common adaptor with T4 DNA ligase.
- Following ligation, the template undergoes PCR using TITANIUM™ Taq DNA polymerase.
- Once the product has been purified and normalized, it is then fragmented with Fragmentation Reagent (DNAse I).
- The fragmented DNA is end-labeled using terminal deoxynucleotidyl transferase.
- The Biotin-labeled products are hybridized to the SNP arrays.
- The hybridized array is stained with streptavidin phycoerythrin conjugate and scanned by the GeneChip® Scanner 3000 DX V.2. The amount of light emitted at 570nm is proportional to the bound target at each location on the array.
The raw data will be generated using Affymetrix software (GCOS). Detailed copy number variation and other chromosomal abnormalities will be identified by using additional software including Partek Genomics Suite.
High-throughput Genotyping Assay "back to top"
Genome-wide association studies and linkage analyses play an increasingly important role in our understanding of the pathogenesis of a wide range of diseases, encompassing immune as well as vascular, neuropsychiatric, metabolic, malignant and gastrointestinal etiologies.
The GeneChip® Human Mapping 500K Arrays provide a powerful tool for high throughput SNP genotyping. It is comprised of two arrays, each capable of genotyping on average 250,000 SNPs (approximately 262,000 for Nsp arrays and 238,000 for Sty arrays). The SNPs on the Arrays were chosen for even distribution across the genome with a mean marker distance of 5.6 kb, providing researchers with more power and confidence to detect association and markers with the diseases of interest. Recently, Affymetrix® has released Genome-Wide Human SNP Array 6.0, which features more than 1.8 million markers for genetic variation, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes for the detection of copy number variation. The Genome-Wide Human SNP Array 6.0 thus provides a robust, flexible, cost-effective approach for scoring SNP genotypes in large numbers of samples and will provide a new technological paradigm for the design of whole-genome SNP-based association studies.
The assay involves the following 7 key steps for 500k arrays:
- 250 ng of genomic DNA is initially digested with the restriction enzymes Nsp I or Sty I.
- Then ligated to a common adaptor with T4 DNA ligase.
- Following ligation, the template undergoes PCR using TITANIUM™ Taq DNA polymerase.
- Once the product has been purified and normalized, it is then fragmented with Fragmentation Reagent (DNAse I).
- The fragmented DNA is end-labeled using terminal deoxynucleotidyl transferase.
- The Biotin-labeled products are hybridized to the SNP arrays.
- The hybridized array is stained with streptavidin phycoerythrin conjugate and scanned by the GeneChip® Scanner 3000 DX V.2. The amount of light emitted at 570nm is proportional to the bound target at each location on the array.
The raw data will be generated using Affymetrix software (GCOS). Detailed analysis will be performed to identify disease-associated markers/genes or chromosomal regions.
