2. Drug – Body Interactions
When we talk about drug targets (direct or indirect), we
not only talk about the gene expression, but also the
mutation and the epigenetic status of these targets. All of
these factors, together with the drug activity itself, affect
the efficacy of a therapeutics.
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Sample & Assay Technologies
3. Today’s Agenda
1. Overview the actively investigated cancer drug target
genes
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.
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2. Profile key cancer-related gene expressions
3. Detect gene mutations
4. Analyze the histone modifications of key cancer drug
target genes
.
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Sample & Assay Technologies
8. 3 Purposes of Using Cancer Drug Targets PCR Array
1. Screen
Profile the key cancer drug target genes before and after
the treatment (drugs, chemicals, siRNA, shRNA,
plasmids…) to identify the targets.
.
.
.
.
.
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2. Validate – After getting the results of microarrays;
We also provide primers for individual
genes.
3. Find other novel co-targets.
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Sample & Assay Technologies
11. Profile Cancer Type-Specific Genes
.
1. Breast Cancer
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-131A.html
.
.
2. Lung Cancer
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-134A.html
.
.
3. Liver Cancer
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-133A.html
.
.
4. Leukemia
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-137A.html
.
.
5. Lymphoma
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-139A.html
.
.
6. Prostate Cancer
http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-135A.html
.
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Sample & Assay Technologies
12. Study Gene Mutations
1. In many cases, cancer drug targets are mutated
genes; or else mutated genes affect cell response to a
specific drug, how to identify these gene mutations?
QIAGEN® works in partnership with Amgen, Merck Serono, and BMS/Lilly/Imclone to
develop global companion diagnostics for detecting KRAS mutations to support the antiEGFR therapies — Vectibix® and Erbitux® —in patients with metastatic colorectal cancer.
2. If you have known a mutant gene as a drug target, is
this mutant gene expressed in your samples?
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Sample & Assay Technologies
13. Principle of Cancer Mutation PCR Arrays – DNA Technology
* Real-time PCR is the most sensitive and reliable method for the detection of DNA
.
mutations.
* Our scientists have developed real-time PCR assays to detect lower than 1% somatic
.
mutations in the background of wild-type DNA templates.
* Amplification Refractory Mutation System (ARMS®) technology is a strategy in which
.
primers with a mismatched 3’-residue will not function as primers in a PCR reaction. In other
words, ARMS-designed primers will preferentially amplify the mutant sequence. The single
nucleotide mutations are selected from comprehensive somatic mutation databases
(COSMIC) and peer-reviewed scientific literature based on their clinical or functional
relevance and frequency of occurrence.
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Sample & Assay Technologies
14. An Example of ARMS®
Template: Wild-Type AGTCAGTC
Primer TCAGTAAA
AGTCAGTC
Won’t Work
.
.
.
Template: Mutation AGTCAGTT
Primer TCAGTAAA
AGTCAGTT
Will Work
.
.
.
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Sample & Assay Technologies
15. Cancer Mutation PCR Array Design
Design:
.
Cancer Mutation PCR Array is a collection of mutation specific
assays for multiple mutations in a single gene, as well as mutations
present in genes in downstream signaling activities.
The mutations are selected from comprehensive databases (such
as the COSMIC database) and literature reviews based on their clinical or
functional relevance and frequency of occurrence.
.
.
Example: EGFR Pathway Somatic Mutation PCR Array – 96 well plate
• EGFR Mutations: 20
• PI3K Mutations: 7
• AKT1 Mutations: 1
• PTEN Mutations: 6
• KRAS Mutations: 16
• HRAS Mutations: 11
• NRAS Mutations: 12
• BRAF Mutations: 8
• MEK1 Mutations: 4
.
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Sample & Assay Technologies
16. Different Cancer Mutation PCR Arrays and Assays –
Designed to Discover and Verify Drug Target Biomarkers
Custom available!
(New!)
We have just added
esophageal cancer
and head & neck
cancer.
We also provide over 900 individual gene assays:
http://www.sabiosciences.com/mutationproductlist.php?tab=browsebygene
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Sample & Assay Technologies
17. Study the Epigenetic Status of the Genes
Histone modifications define chromatin structure, and
correlate closely with the transcriptional activity of the
associated genes.
.
Adding HDAC (histone deacetylase) inhibitors to a drug
regimen can greatly change the cell response (e.g. The 4
.
FDA-approved epigenetic drugs for clinical use include 2 DNA demethylating agents, 5azacytidine and decitabine, and 2 histone deacetylase (HDAC) inhibitors, vorinostat and
valproic acid. So far, epigenetic drugs have been approved mainly for the treatment of blood
cancers).
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.
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Sample & Assay Technologies
18. Introduction to Histone Modification
Five major families of histone exist: H1, H2A, H2B, H3, H4 and H5.
Histones H2A, H2B, H3 and H4 are known as the core histones, while
histones H1 and H5 are known as the linker histones.
.
Major modifications of histone include acetylation, methylation,
phosphorylation, ubiquitination, and sumoylation. Combinations of
histone modifications constitute a code, the so-called “histone
code”.
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H3K4me1 means the monomethylation of the 4th residue (a lysine) from the start (i.e., the Nterminal) of the histone H3 protein.
.
The common nomenclature of histone modifications is:
1. The name of the histone (e.g., H3)
2. The single-letter amino acid abbreviation (e.g., K for lysine) and the amino acid position
in the protein
3. The type of modification (me: methyl, p: phosphate, ac: acetyl, ub: ubiquitin)
.
.
.
.
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Sample & Assay Technologies
19. An Example Experiment with ChIP PCR Array
Together with our easy and fast EpiTect One-Day ChIP Kit
(http://www.sabiosciences.com/chipqpcr_product/HTML/334471.html), ChIP-Grade Antibody
Kit (http://www.sabiosciences.com/chipgradeantibody.php), one million cells per assay as
starting material, ChIP PCR Arrays provide 100% effective call rates.
.
.
P19 mouse embryonic carcinoma cells were prepared for ChIP Assay using the EpiTect OneDay ChIP Kit and anti-H3K4me3 Antibody Kit. One million cells were used as starting material
for each ChIP Assay. The purified ChIP DNA samples were characterized using Mouse Stem
Cell Transcription Factor ChIP PCR Array with 1/100th of the ChIP DNA as template in each
well. The Real-Time PCR results demonstrate 100 % effective call rates for the Input Fraction
(Ct < 30). The difference of Ct value between the anti-H3K4me3 antibody and the control IgG
fractions indicates the specific enrichment of the antibody, whereas the high Ct value of the
control IgG fraction indicates the low background of the assay.
.
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Sample & Assay Technologies
21. Other ChIP PCR Arrays
Please Note: Our cataloged ChIP PCR Arrays
(http://www.sabiosciences.com/chipqpcrarrays.php) are for
histone modification study only. If you are interested in transcription factor
binding sites or other nuclear factor binding sites, please order
custom-designed ChIP PCR Arrays from us.
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Sample & Assay Technologies
22. We Provide Service – Send Samples to Us & Receive Results!
Whole Genome
Illumina Gene Expression Profiling
Illumina Genotyping
.
Pathway / Focused Panel
PCR Array
miRNA PCR Array (special miRNome Profiling service for US customers in May!)
http://www.sabiosciences.com/promotion/servicecoremirnapromo.php
Mutation Profiling
Methylation
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Individual Gene / Locus
Real-time PCR
Mutation Detection
Methylation
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Sample Preparation – DNA, RNA Extraction and Purification
Cells, Tissue or Biofluids
Fixed Tissue
Small Sample
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Sample & Assay Technologies