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Research essay by Kedar Ghimire/ Jacobs University Bremen
 
"Microarrays”
Application and potentialSubmitted by
Kedar Ghimire,Earth
Jacobs University Bremen9/5/2007
CourseHigh-throughput Screening Technology II/ Instructor of record: Dr. Helge Weingart
 
Research essay by Kedar Ghimire/ Jacobs University Bremen
 Introduction
The central dogma of biology suggests us that the genetic information flows fromDNA to RNA and finally to the translation of proteins. This theory was first proposed byFrancis Crick in 1957. The DNA gets coded to mRNA (messenger RNA) by the processcalled transcription. This process is facilitated by RNA polymerase and transcriptionfactors. RNA structure resembles with that of DNA in many aspects except that RNA issingle stranded and the DNA is double stranded and also has uracil instead of thymine inDNA. This mature mRNA will be transported into ribosome where it will be translatedinto amino acids. A series of 3 nucleotides determines the coding one amino acid.Eukaryotes genes contain coding regions (exons) and non-coding regions(introns). RNA goes through a series of modifications where exons are joined. Theintrons are removed by post-transcriptional modifications. This RNA formed after removing the non coding region is now termed as the mRNA. The process fromtranscription to translation can be referred to as gene expression. Epigenetics,environmental factors, cellular developmental stages are critical parameters guiding thegene expression pattern. One of the consequences of central dogma is that the genescodes for proteins. In order to study the expression pattern of thousands of genes we needa technology which is relatively convenient and can give a faster and reliable high-throughput. Older technologies to study gene expression pattern were often tedious andtime consuming. Hence Microarrays has proven to be a revolution to molecular biologistin studying the gene expression.Research in molecular biology increased its scope in recent decades from the needof monitoring expression level of few genes to thousands of genes. By the help of thistechnique a gene can be monitored at its transcription level. Expression data of a mRNAfrom a large pool of genes can be easily collected by the help of DNA microarrays. DNAMicroarray technology can be simply defined as a recent development that helps to knowhow much mRNA corresponding to a particular cell is present. This technique is a resultof the technological advancement in the field of micro-fluidics, robotics and computer technology. Human Genome Project (HGP), Polymerase Chain reaction (PCR) and the base pairing rule found by Watson and Crick are some of the important groundwork which led to the development of this gene technology.
 Today, we can getthousand-fold high sensitivity in just a few minutes using modernmicroarray slides, fluorescent dye probes and laser detectors. Whatonce used to take two days to visualize on X-ray film using radioactivetest DNA now takes only few minutes, and also is automatically savedas a computer file. Microarray has revolutionized the field of lifescience today.
Various types of microarrays are in development like protein microarray, antibodymicroarray, tissue microarray but this article would focus on DNA microarray.Complementary DNA Microarray (cDNA Microarrays) and oligonucleotide microarraysare two common types of microarrays used for gene expression. The main difference between these two is in their experimental design. C-DNA which can be obtained from
CourseHigh-throughput Screening Technology II/ Instructor of record: Dr. Helge Weingart
 
Research essay by Kedar Ghimire/ Jacobs University BremenmRNA can be used as targets and upon fluorescent labeling they will be hybridized ontothe Microarray.In order to understand the basic principle, a very simple application is provided.Suppose we are interested in studying whether a particular cell expresses the proteinmyoglobin. In order to achieve this we should isolate mRNA from the cell under observation. This single stranded RNA can be made to bind with the DNA probes that arein the chip surface. Finally a fluorescent dye can be used to study whether a particular DNA in the DNA chip has hybridized with the RNA or not.Suppose if we have following mRNA sequence available:
5' GAGCAAGCAU CCCGGGACU UUGGUGCUGA UGCCAGGGG 3'
Then the correct DNA target would be:
3’CTCGTTCGTA GGGCCCCTGA AACCACGAC ACGGGTCCCC5’
From the above mentioned sequences RNA-DNA hybrid, the following of the doublestranded hybrid is made:5' GAGCAAGCAU CCCGGGACU UUGGUGCUGA UGCCAGGGG 3'3’ CTCGTTCGTA GGGCCCCTGA AACCACGAC ACGGGTCCCC 5’In this particular example, we do not see a great advantage of this DNA chip because we are looking at only a single gene. But if we were to use older techniques for 100000 genes then we had to carry the experiment 100000 times. But if we use a DNAchip then we can have 100000 different probes where the single stranded RNA canhybridize with DNA probes. One good advantage of DNA chip is that we can reuse it.
 Design of DNA chips
The design of DNA chips is usually divided into three different phases:
Step 1: Designing Gene Probes
A known sequence of DNA strands can be made by using Polymerase chainreaction (PCR). This known sequence acts as a probe to be hybridized with the singlestranded RNA from the sample under investigation.Probes can be generated by two different methods. One of the methods uses PCR to generate cDNA probes. The other method utilizes synthetic oligonucleotide generated by chemical methods and spotted onto the chip by using photolithography. This latter approach is widely used by companies such as Affymetrix and Agilent Chips.While other companies like Nanogen Inc. use their own particular techniques to prepare probes and arrays and a lot of varieties are thus available according to the need toresearch and experiment.
CourseHigh-throughput Screening Technology II/ Instructor of record: Dr. Helge Weingart
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