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