Monday, October 19, 2009

About us



We are students of Universiti Teknologi Malaysia and taking Bachelor of Science (Industrial Biology). Industrial biology is the application of scientific and engineering principles to the processing of materials by microorganisms or plant and animal cells to create novel products or processes. Industrial biology which is based on Biotechnology is one of the key technologies that helps Malaysia to transform into highly developed nation by the year 2020. It is the technology of the milennium which can ensure fooe security, better healthcare, cleaner and safer environment.

After the ICT revolution, biotechnology is generally regarded as the next engine of economic growth. The degree in industrial biology is a 3-year programme which integrates various fields of biological sciences for instance, microbiology, biochemistry, molecular genetics, bioinformatics, fermentation technology, plant and animal cell culture, environment, nanobiotechnology, genetic engineering and bioprocess engineering. In this course, student will be trained to apply high technology knowledge to solve current biological science problem related to biotechnology.

This webpage is mainly made for our assignment in the subject of Technique of Molecular Biology. In this assignment, we need to choose a technique that widely used in molecular biology and explained about it. We are keen to know more about DNA microarray, thus we make this technique as the title for our assignment. The information includes in this webpage is the collection of our readings and knowledge from scientific journal and other webpage.

We hope that these information will help you to understand better about DNA microarray and anything related with it. Believe us, that DNA microarray is a very useful technique in recent development of molecular biology. We hope that readers will enjoy reading the information given.

thank you..

Contact Us

Nur Hidayah binti Junaidi
AS070393
880925-01-5422
hidayah.junaidi@gmail.com



Nurul Ainun binti C. Abllah
AS070435
880309-11-5366


Ruqaiyah Bevee binti Rizal

AS070498

870315-12-5326

rbevee2@rocketmail.com

Introduction


A DNA microarray is a solid surface (glass slide or silicon chip) consisting of an arrayed series of thousands of microscopic spots of specific DNA sequences, each representing a gene or other DNA element. These spots are known as probes. The array hybridized to fluorescently labeled DNA samples known as the target. Probe-target hybridization is detected and quantified to determine the relative abundances of specific nucleic acid sequences in the target. Since an array can contain a whole genome of an organism, it is a tool of major importance for genome-wide studies (Yongtau, 2009). Some of usefulness of DNA microarrays are:


Can follow the activity of many genes at the same time.
Can get a lot of results fast.
Can compare the activity of many genes in diseased and healthy cells.
Can categorize diseases into subgroups.

DNA microarray has several significant advantages which are highly parallelism, highly automation and miniaturization. In 1994, the first commercial gene chip was released by Affymetrix Inc. In 1995, the first DNA microarray application was reported by Schena et al. Microarrays can be fabricated by using a variety of technologies, including printing with fine-pointed pins onto glass slides (spotting), photolithography using premade masks, photolithography using dynamic micromirror devices, ink-jet printing and soft lithography also known as molecular stamp (in-situ synthesis). Microarrays fabricated by in-situ synthesis provide higher density, better reproducibility and little batch to batch variation than those fabricated by spotting technique, but much higher cost (Teng and Xiao, 2009).

In spotted microarrays, the probes are oligonucleotides, cDNA or PCR products. The probes are synthesized and then “spotted” onto the glass slide. The matrix of probes represents the nucleic acid profiles of the prepared probes and is ready to capture complementary DNA or cRNA targets derived from experimental or clinical samples. This technique is used to produce “in-house” microarrays according to the different experimental design easily. This provides a relatively low cost microarray that may be customized for each study, and avoids the costs of purchasing expensive commercial microarrays. The first microarrayer was assembled by Shalon, Brwon and others from Stanfrod University. Other commercial models were also available from different manufacturers such as Cartesian Technologies, TeleChem, Biodot, PerkinElmer, GE Healthcare in United States, Genetix Corporation in the United Kingdom and Capital Bio in China (Teng and Xiao, 2009).

Types of Array

Spotted DNA array

Developed by Pat Browns Lab at Stanford
Usually has to make your own.
Relatively cheap
Flexible( can spot anything you want)
Less accurate in extreme of range
Cheap so that can repeat experiments many times.
Highly variable spot deposition.

Affymetrix or gene chips

Limited types
Expensive
Dynamics range may be slightly better in results.
Fewer repeated experiments
More uniform DNA testing

Ink-jet/Bubble Jet Microarrays

Deposition of cDNA/oligos
Highly uniform spots
In situ synthesis
Building 25-60 mer oligos directly on arrays
Flexible
Good for protyping

Application


At present, there are two main trends in DNA microarray fabrication. The first one is high-density of microarray that more than 1 million probes can be mounted onto every square cm by in situ synthesis. This might help the researches to manifest the whole genome analysis of one species on a single microarray. The second one is minimization of reaction systems, which refers to the minimization of required testing samples of the microarray. The minimum starting requirement of total RNA or DNA for DNA microarray hybridization is of nanogram level, which makes it possible to study the expression profile of a single cell. This is especially useful in iPSCs (induced pluripotent stem cells) research field.

A study using DNA microarray technology was carried out by Friend and colleagues in 2002, who provide a good example of the use of expression profiling by DNA microarray to improve breast cancer classification and therapy. Many breast-cancer patients receive unnecessary chemotherapy or hormonal treatment for possible tumor spread after removal of a primary tumor. Classical histopathological and clinical criteria are insufficient to determine which patients need this adjuvant treatment.

Friend and colleagues analysed primary breast tumor from a mix of patients who developed metastases or remained disease-free after 5 years, using DNA microarrays to study the gene expression profiles. They were able to identify 70 genes significantly associated with disease outcome, and developed a prognosis classifier based on this microarray study. This constituted a powerful tool for tailoring treatment to those patients at risk of recurring disease, and avoiding unnecessary treatment and the associated costs in patient quality of life and health-care expenditure.

Yongtao Xue-Franzen. 2009. DNA microarray approaches to understanding the regulation and evolution of gene expression networks. Stockholm, Sweden. Karolinka University Press

HAN ZeGuang’s team from the National Human Genome Center at Shanghai reported on a comprehensive characterization of gene expression profiles of hepatitis B virus-positive hepatocelluar carcinoma through a cDNA microarray containing 12393 genes/ESTs. Integrated data identified 2253 genes/ESTs as candidates with differential expression. A number of genes related to oncogenesis and hepatic function/differentiation were selected for further study. The altered transcriptome profiles in hepatocelluar carcinoma could be correlated to a number of chromosome regions with amplification or loss of heterozygosity, providing one of the underlying causes of the transcription anomaly of hepatocelluar carcinoma. In 2002, ZHANG Xu’s team from Institute of Neuroscience, Chinese Academy of Sciences used the gene expression microarray to study the gene expression of dorsal root ganglia in the rat peripheral nerve injury model. They tried to elucidate the gene expression profile by using a cDNA microarray with 7523 genes and found a group of genes related to nerve injury and pain which could be considered as potential drug targets. The results also suggested the molecular mechanisms of Gabapentin engaged in the clinical therapy for pain

Gunderson et al. utilized DNA microarray to detect SNP locus in human genome. These data showed high consistence with the traditionally PCR-based technique. Amplichip CYP450 testing chip made by Roche was approved by FDA to enter the clinical trials in 2005. This chip could be used to analyze polymorphism loci that may determine the activity of cytochrome oxidase and predict whether the drug metabolism rate is high or low in patients. That was the first SNP microarray used in clinical. Later on, Burton et al. examined 14500 SNP loci between autoimmunity disease patients and healthy individuals by microarray. The study included 1000 patients suffering four diseases and 1500 health individuals as the controls

In 2006, researchers from Chinese National Human Genome Center and National Engineering Center for Biochip at Shanghai analyzed the relevance between genome copy number alterations and transcriptional expression of hepatitis B virus associated with human hepatocellular carcinoma through comparative genomic hybridization (aCGH) and gene expression profiling approaches jointly. This work provided new insights into the novel genetic mechanisms in hepatocarcinogenesis associated with hepatitis B virus. Carter et al. reported that up to 12% of the human genome and thousands of genes are variable in copy number. This diversity is likely to be responsible for a significant proportion of normal phenotypic variation. The discovery is far beyond the forecast ever before. Lee et al. reported that mental retardation was genotyped using comparative genomic hybridization (aCGH). One deletion of chromosome region 15q13.3 was found in the patients, and the work was published in Nature Genetics.

X. K. Teng., H.S. Xiao., 2009. Perspectives of DNA microarray and next-generation DNA sequencing technologies. Sci China Ser C-Life Sci 52 (1): 7-16

Development

In-situ synthesis was first applied by Fodor and his colleagues to produce a DNA microarray. Affymetrix Inc developed photolithography (Fodor et al, 1991) in-situ synthesis method, which uses photo-chemical protection to synthesize oligonucleotide probes on the substrate directly. The advantage of this method is high accuracy while the drawback is time-consuming and expensive in manufacturing optical masking agents. Singh-Easson’s research group from University of Wisconsin used the computer designed virtual mask also known as photolithography using dynamic micro-mirror devices to prepare DNA microarrays.

An optical digital micro-mirror device, including 480000 small aluminum mirror arrays, was used to precisely control the direction of light reflection in the mirror by the computer, beaming the ultra-violet radiation directly to the glass substrate for selective removal of the protection of the base in a particular region of the substrate. Virtual mask technology is cost-effective and less time-consuming (Singh-Gasson et al, 1999). Nimblegen Company has taken advantage of this technology in production of the high density commercial DNA microarrays (Teng and Xiao, 2009).

LU Zuhong’s group (Xiao et al, 2002) from Southeast University in China has established another in-situ synthesis of oligonucleotide microarrays on glass surfaces by using soft lithography (called molecular stamp). This method is based on the standard phosphoramidite chemistry protocol. The coupling was achieved by the glass slide being printed with a set of polydimethylsiloxane (PDMS) microstamps, on which spread nucleoside monomer and tetrazole mixed solution. The elastic characteristic of PDMS allowed it to make conformal contact with the glass slide in the printing coupling. In this way, oligonucleotide microarrays can be fabricated with a feature size of 100 μm and 30 μm. In China, the government initiated the projects for research and development of biochip technology by the end of 1990s.

The first DNA microarray in China was made under the financial support from the Human Genome Project of the Chinese Academy of Sciences, using a large number of human cDNA clones identified by EST sequencing conducted by Chinese National Human Genome Center at Shanghai. It was successfully used in the first case of transcriptome comparison of hepatocellular carcinoma with those of the corresponding noncancerous tissue. This work was published in the PNAS in 2001 (Xu et al, 2001) which represent the breakthrough of DNA microarray application in China. By the end of the Ninth Five-Year Plan, research groups in Beijing, Shanghai and Nanjing began to accelerate the biochip technology study owing to the strategic support from the National High-Tech Developing Programs (also known as “863” Program). Hua Guan Biochip Company in Shanghai was thus set up and engaged mainly in research and development of diagnostic biochip (Teng and Xiao, 2009).

In addition, a number of domestic private enterprises have involved in the research and development of biochips, such as “Yi-sheng Tang” in Guangzhou, “Bio-Star” in Shanghai and “JianNan Biotechnology” in Zhejiang. In 2000, the government has invested nearly 500 million RMB in succession to set up two national engineering research centers for development of biochip technology and products in Beijing and Shanghai.

Under the support of “863” and other national grants during the Tenth Five-Year Plan and the Eleventh Five- Year Plan, a series of biochip technologies and products have been developed and commercialized. For example, the National Engineering Center for Biochip at Beijing has developed DNA microarray scanners, microarrayer, hybridization oven and sample handling equipments, forming a complete set of instruments for fabricating and application of DNA microarrays (Teng and Xiao, 2009).

Meanwhile, commercial products including gene expression microarrays, mRNA microarrays, CGH microarrays, DNA methylation microarrays have been developed through their own platform, which represents the establishment of a complete industry chain for fabrication and application of DNA microarrays with independent intellectual property rights. Microarrays start to be applied in food safety examination and clinical diagnosis.

The National Engineering Center for Biochip at Shanghai, on the other hand, established a DNA microarray platform based on the printing technology and developed independently more than 20 kinds of gene expression microarrays and pathway microarrays, including the genome-wide expression microarrays of rats, mice and microorganisms. At the same time, the globally widely used systems, such as Affymetrix, Agilent, Illumina and Nimgblegen, were also introduced into China to provide comprehensive solutions of microarray application (Teng and Xiao, 2009).

A standard operation protocol (SOP) and the criteria for quality controls were established which in turn promote the development of DNA microarray in China. Until now, more than 100 research papers have been published in PNAS (Zheng et al, 2005; Huang et l, 2006), Genomics (Hua et al, 2008; Zhang et al, 2007), BBRC (Zhao et al, 2007; Li et al, 2006), JBC (Ma et al, 2006), FEBS Letter (Huang et al, 2006; Xu et al, 2005), and other international journals related to DNA microarray application supported by “973”, “863”, “NSFC” and Shanghai Science and Technology Commission (Teng and Xiao, 2009).

References

Yongtau Xue-Franzen (2009). DNA Microarray Approaches to Understanding The Regulation and Evolution of Gene Expression Networks. School of Life Sciences, Sodertorn University, Huddinge, Sweden.

Teng, X. K., Xiao, H. S. (2009). Perspectives of DNA microarray and next-generation DNA sequencing technologies. Science in China Series C: Life Sciences, 1-3).

Schena, M., Shalon, D., Davis, R. W., Brown, P.O. (1995). Quantitative Monitoring of Gene Expression Pattern with a Complementary DN

A Microarray. Science, 270, 467-470.

Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler, J., Lockhard, D. J., Morris, M. S., and Fodor, S. P. (1996). Accessing Genetic Information with High-density DNA Arrays. Science, 274, 610-614.

Lashkari, D. A., DeRisi, J. L., McCusker, J. H., Namath, A. F., Gentile, C., Hwang, S. Y., Brown, P. O. and Davis, R. W. (1997). Yeast Microarray for Genome Wide Parallel Genetic and Gene Expression Analysis. Proc Natl Acad Sci USA. 94, 13057-13062.

Fodor, S. P., Read, J.L., Pirrung, M. C. et al. (1991). Light Directed, Spatial Addressable Parallel Chemical Synthesis. Science. 251(4995), 767-773.

Singh-Gasson, S., Green, R. D., Yue, Y. et al. (1999). Maskless Fabrication of Light-directed Oligonucleotide Microarrays Using Digital Micromirror Array. Nat Biotechnol. 17(10), 974-978.

Zheng, P. Z., Wang, K. K., Zhang, Q. Y. et al. Systems Analysis of Transcriptome and Proteome in Retinoic Acid/Arsenic Trioxide-induced Cell Differentiation/Apoptosis Leukimia. Proc Natl Acad Sci USA. 102(21), 7653-7658

Huang, W., He, Y., Wang, H. et al. (2006). Linkage Disequilibrium Sharing and Haplotype-tagged SNP Portability between Populations. Proc Natl Acad Sci USA. 103(5), 1418-1421.

Hua, Y. J., Tu, K., Tang, Z. Y. et al. (2008). Comparison of Normalization Methods with Microarray RNA Microarray. Genomics. 92(2), 122-128.

Sunday, October 18, 2009

Types of DNA Microarray

Microarrays experiments has been classified into three basic types based on the sample which the two types grouped in genomics while the others is “transcriptomic”, means that this kind of array measure the level of mRNA. The different between this microarray are the types of immobilized DNA used to generate the arrays and also the different kind of information or results obtained from chips.

Types of transcriptomic

Changes in Gene Expression Levels

In this case, determination level or volume of certain gene expression called “microarrays expression analysis” and the arrays used in this types called “ expression chips” .The immobilized DNA used is cDNA derived from mRNA of known gene. The sample and control cDNA hybridize to the chips derived from normal and diseased tissue. If the sample cDNA (derived from diseased tissue) is over expressed than the normal cDNA, then when fluoresce applied to chips, its show that there will be more red fluoresce intensity than the green one. This kind of microarrays used to drug development, drug responds, therapy development and disease diagnosis. For examples certain genes is over expression in a particular form of cancer, then researchers can use expression chips to see a new drugs that would reduce over expression and force the cancer into remission.


Types of genomics

Genomic Gains and Losses

Researchers use a technique called microarray Comparative Genomic Hybridization (CGH) to look for genomics gain or loses in certain types of disease gene or for a changes in copy number of particular gene involved in diseased. In microarray CGH, the large pieces of genomics DNA is our target and in the hybridization mixture contain fluorescently labeled DNA obtained from normal (control) and diseased tissue. Therefore, if the copy of target gene has increased, the sample DNA would hybridize on the spot greater than control DNA o n the spot. As a results, spot containing high intensity of red fluoresce then green fluoresce and this showed that the copy number of diseased number has increased. This kind of arrays used in tumor classification, risk assessment, prognosis prediction,

Mutations in DNA

This types of mutation microarrays analysis is used by researchers to detect mutation or polymorphisms in gene sequences,the target or immobilized DNA. In this case, the target gene placed in given spot within the arrays but differs from other spots in same microarrays by put one or a few specific nucleotide. In this analysis the immobilize DNA is sequence known as Single Nucleotide Polymorphisms or SNP (a small genetics changes in a person DNA sequences). These microarrays different from the expression and CGH microarrays because it only needs DNA control samples in hybridization solution. When genomic DNA from a individual is hybridized to an array loaded with SNPs, then the sample would only hybrid to its specific SNPs of that person and the fluoresce applied showed that the color intensity indicate that person either in risk or not.


History

A DNA microarrays is a solid surface (glass slide or silicon chips)

1988: First patents applied for DNA microarrays


1991
: Photolithographic printing (Affymetrix), first licensing of DNA microarray patents granted


1994
: First cDNA collections are developed at Stranford, Eupropean Patent Specification (EP0373203) for DNA microarrays granted


1995
: Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Oxford Gene Technology Limited founded by Professor Edwin Southern to exploit patented technology developed in his research laboratories at Oxford University


1996
: Commercialization of arrays (Affymetrix)


1997
: Genome- wide expression monitoring in S. cerevisiae (yeast), First US patent (US5700637) for DNA microarrays granted, Microarray patents assigned from Oxford University (Isis) to OGT


2000
: Portraits/ Signatures of cancer.


2003
: Introduction into clinical practices


2004
: Whole human genome on one microarray


Before the era of DNA microarray technology, Differential Display Technology (Liang and Pardee, 1992) was a major tool that opened the door to interpretation of genome-wide information. This technology is allows detection of altered gene expression patterns by running a DNA sequencing gel after PCR amplification of total mRNA samples from control and experimental conditions. The popularity of this technology did not last long due to it high false positive rate and the impossibility of identifying more than a few genes. This “differential sequencing” was replaced by differential hybridization within a few years. This technique relied on the “reverse northern” hybridization approach: known cDNA probes are spotted on nylon membranes and hybridized by control and experimental samples. This eventually led to the development of currently popular technology, DNA microarray, pioneered by Patrick Brown and David Botstein in 1995 (Schena et al, 1995) and Chee in 1996 (Chee et al, 1996). In 1997, a complete eukaryotic genome (Saccharomyces cerevisiae) was established on a microarray (Lashkaari et al, 1997).