Post on 27-Jan-2015
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Microarray - CGHPinal
Chaudhari
Contents… Introduction.
Microarray and microarray CGH Timeline of recent DNA Microarray developments. Designing a microarray experiment.Basic steps. Applications Advantages Limitations References
Microarray …Introducti
on;A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated.
By using an array containing many DNA samples, scientists can determine, in a single experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array.
With the aid of a computer, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell.
Background on DNA microarrays.…The basic premise of the DNA microarray is
that RNA samples or targets are hybridized to known cDNAs/oligo probes on the arrays.
Microarrays were originally designed to measure gene expression levels of a few genes. Recently, high density microarrays have been developed which have allowed the global analysis of gene expression or the transcriptome.
This global analysis allows one to determine the cellular function of genes, the nature and regulation of biochemical pathways, and the regulatory mechanisms at play during certain signalling conditions or diseases
What exactly is a Microarray …DNA Microarrays are small, solid supports onto
which the sequences from thousands of different genes are immobilized, or attached, at fixed locations.
The supports themselves are usually glass microscope slides, the size of two side-by-side pinky fingers, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support.
It is important that the gene sequences in a microarray are attached to their support in an orderly or fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides.
• Timeline of Recent DNA Microarray Developments
• 1991: Photolithographic printing (Affymetrix)
• 1994: First cDNA collections are deve;oped at Stranford
• 1995: Quantitative monitoring of gene expression patterns with a complementary DNA microarray.
• 1996: Commercialization of arrays (Affymetrix)
• 1997: Genome- wide expression monitoring in S. cerevisiae (yeast)
• 2000: Portraits/ Signatures of cancer.• 2003: Introduction into clinical practices• 2004: Whole human genome on one
microarray
Timeline of Recent DNA Microarray Developments.…
Microarray CGH…Introducti
on;Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Array comparative genomic hybridization (also CMA, Chromosomal Microarray Analysis, Microarray-based comparative genomic hybridization, array CGH, aCGH, or Virtual Karyotype) is a technique to detect genomic copy number variations at a higher resolution level than chromosome-based comparative genomic hybridization (CGH).Dedicated tools are needed to analyse the results of such experiments, which include appropriate visualisation, and to take into consideration the physical relation in the genome between the probes on the array.
• Comparative genomic hybridization (CGH) microarray is an emerging tool in bioclinical research that allows to identify genomic alterations with a higher resolution than the conventional CGH
• To study aberrations of the genome, investigators competitively hybridize fluorescein-labeled normal and pathological samples to an array containing clones designed to cover certain areas of the genome.
• Once hybridization has been performed, the signal intensities of the dyes are quantified. Thus, this technique provides a means to quantitatively measure DNA copy-number changes and to map them directly onto a genomic sequence. In oncology, where carcinogenesis is associated with complex chromosomic alterations, CGH arrays can be used for detailed analysis of genomic changes in copy number (in terms of gains or loss of genetic information) in the tumor sample.
Designing a microarray experiment
Basic steps .… 1.Obtain DNA Microarray chip.2. RNA Extraction from samples.3.Cy3 & Cy5 labeling of RNA using (RT-PCR) c-DNA synthesis.4.Hybridization with c-DNA probes to DNA Array.5. Detection of hybridization signal.6.DNA Microarray Data Analysis.
1.Obtain DNA Microarray chip.…
• Manufacturing of MicroarraysFor microarray production, two different
approaches are used:• 1. Synthesis on the chip; and• 2. bulk synthesis with subsequent
deposition on the chip. The first method is applicable to
generate chemical libraries, for example, of short oligonucleotides or peptides; the second method may also be adapted to long polynucleotides or proteins, or any of the many receptors.
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. . . . . . .
Spotting technique.
Nimblegen Maskless Array Synthesis
These "virtual masks" reflect the desired pattern of UV light with individually addressable aluminum mirrors controlled by the computer. The DMD controls the pattern of UV light projected on the microscope slide in the reaction chamber, which is coupled to the DNA synthesizer. The UV light selectively cleaves a UV-labile protecting group at the precise location where the next nucleotide will be coupled. The patterns are coordinated with the DNA synthesis chemistry in a parallel, combinatorial manner such that 385,000 to 2.1 million unique probe features are synthesized in a single array.
RNARNA
2. RNA (DNA)Extraction from samples..
NORMAL CELLS CANCER CELLS
mRNAtRNArRNA
TTTTTT
TA
mRNA
WASHING SOLUTION
tRNArRNA
ELUTION BUFFER
Bead containing poly T chain
mRNA
U AGCAG
TACGTC
mRNA
cDNA
mRNA
cDNA
Normal cells.
3.Cy3 & Cy5 labeling of RNA using (RT-PCR) c-DNA
synthesis.…
ATGCAG
A A
T T
A A
U AGCAG
TACGTC
ATGCAG
A A
T T
A A
Cancer cells.
Array CGH
Test DNA (cy3)
Array containingprobes corresponding togenomic DNA
Scan and analyze image
Mix and hybridize to array
Reference DNA (cy5)
4. Hybridization with c-DNA probes to DNA Array..…
Gene 1 Gene 2 Gene 3
4. Hybridization with c-DNA probes to DNA Array..…
5. Detection of hybridization signal..…
6.DNA Microarray Data Analysis...…
The colors of microarray.…
In this schematic: GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. Each spot on an array is associated with a particular gene. Each color in an array represents either healthy (control) or diseased (sample) tissue. Depending on the type of array used, the location and intensity of a color will tell us whether the gene, or mutation, is present in either the control and/or sample DNA. It will also provide an estimate of the expression level of the gene(s) in the sample and control DNA.
Applications of DNA Microarrays.…
DNA microarrays have been used to examine the gene expression changes under diseases such as cancer. Tumour profiling using DNA microarrays allows the analysis of the development and the progression of such complex diseases. Using DNA microarrays, one can examine targets for drug discovery and potential diagnostic and prognostic biomarkers for many complex diseases.DNA microarrays are used commonly to detect viruses and other pathogens from blood samples and thus can be used as a pathogen detection method.DNA microarrays have been more recently used to identify inheritable markers, and therefore have been used as a genotyping tool.SNP chips based on DNA microarray technology have allowed the high throughput profiling of single nucleotide polymorphisms using a chip or array approach. This has allowed polymorphisms to be more quickly assayed and also their relavence to disease to be easily determined.
Advantages of DNA Microarrays.…DNA microarrays are better than
other profiling methods (such as SAGE, SH, PCR methods) in that they are:
Easier to use
Are high-throughput (can analyze thousands of genes or markers at a time)
Generate large amounts of data in little time
Do not require large-scale sequencing
- Allow the quantitation of thousands of genes from many samples
• High-throughput – Identify: candidate genes, patterns
• Compare two different populations– wild type vs. evolved– normal tissue vs. cancerous tissue• Studing specific chromosomal regions.
Advantages of CGH.…
•Parental diagnosis•Disease diagnosis like cancer•Gene expression•Chromosomal abberations.•Determines gain and loss of chromosomal regions
• Do not necessarily reflect true levels of proteins - protein levels are regulated by translation initiation & degradation as well
• Generally, do not “prove” new biology - simply suggest genes involved in a process, a hypothesis that will require traditional experimental verification
• Expensive! $20-$100K to make your own / buy enough to get publishable data
Limitations of array.…
References.…Identification of disease genes by whole genome CGH arraysLisenka E.L.M. Vissers, Joris A. Veltman*, Ad Geurts van Kessel and Han G. Brunner Department of Human Genetics<http://www.nimblegen.com/><http://www.affymetrix.com/><http://smd.stanford.edu/resources/resinfshtml>
Validation of Sequence-Optimized 70-base Oligonucleotides for Use on DNA Microarrays (http://www.westburg.nl/download/arrayposter.pdf). By John Ten Bosch, Chris Seidel, Sajeev Batra, Hugh Lam, Nico Tuason, Sepp Saljoughi, and Robert Saul.Assessment of the specificity and Sensitivity of the oligonucleotides (50 mer ) microarrays. By Dr. Susanne Schröder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul 1 WG Biotech AG, Microarray Development1 and Department of Bioinformatics2.
50 nucleotide long probes on microarrays enable high signal intensity and high specificity. By Dr. Susanne Schröder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul1 MWG Biotech AG,microarray Development1and Department of Bioinformatics 2, Anzinger Str. 7, 85560 Ebersberg, Germany.Optimization of Oligonucleotide - based DNA microarrays. By Angela Relogio, Christian Aschwager, Alexandra Ritcher, Wilhelm Ansorge and Juan Valcarel.
An experimental loop design for the detection of constitutional chromosomal aberrations by array CGHby: Joke Allemeersch, Steven Van Vooren, Femke Hannes, Bart De Moor, Joris Vermeesch, Yves Moreau
Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays Michael D. Kane, Timothy A. Jatkoe, Craig R. Stumpf, Jia Lu1, Jeffrey D. Thomas and Steven J. Madore*
DNA Microarrays: Background, InteractiveDatabases, and Hands-on Data Analysis A. Malcolm Campbell1 and Laurie J. Heyer2
Microarray CGHBen Beheshti, Paul C. Park, Ilan Braude, and Jeremy A. Squire
http/www.wikipedia.com
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