Gctaagccgta cDna Cgattcggcat Oligonucleotide probe

Rinse off unhybridized cDNA

Rinse off unhybridized cDNA

Scan with laser scanner

Fig. 12.13 The application of microarrays or the study of viral products produced in an HSV-infected cell. A DNA microarray for analysis of mRNA in HSV-infected cells. Oligonucleotides complementary to each viral transcript are bound to a glass slide along with oligonucleotides complementary to a number of diagnostic cellular transcripts. Samples of mRNA are isolated from cells under different conditions of infection, and cDNA copies are made using a dye-substituted deoxy-base; a different fluorescent dye is used for each condition. Then the cDNA is hybridized to the chip, unhybridized material washed away, and bound material is localized by scanning with a laser of a wavelength that only excites one or the other of the two dyes. The color and intensity of the signal in each spot can then be directly related to the amount of mRNA present in the original sample.

Intensity is measure of amount

Scan with laser scanner

Fig. 12.13 The application of microarrays or the study of viral products produced in an HSV-infected cell. A DNA microarray for analysis of mRNA in HSV-infected cells. Oligonucleotides complementary to each viral transcript are bound to a glass slide along with oligonucleotides complementary to a number of diagnostic cellular transcripts. Samples of mRNA are isolated from cells under different conditions of infection, and cDNA copies are made using a dye-substituted deoxy-base; a different fluorescent dye is used for each condition. Then the cDNA is hybridized to the chip, unhybridized material washed away, and bound material is localized by scanning with a laser of a wavelength that only excites one or the other of the two dyes. The color and intensity of the signal in each spot can then be directly related to the amount of mRNA present in the original sample.

bind to a subset of the experimental mix. The volume of the experimental solution is kept very small by doing the incubation in a very small chamber. For example, if the probes were bound to a glass slide, layering a glass coverslip over the entire array could form the incubation chamber. Obviously, the tighter the patterning of spots in the array, the smaller the total volume needed. Ideally, a solution of materials from a few or even a single individual infected cell could be the source.

Following incubation and rinsing, interactions between chip probes and the experimental mixture added can be assayed by laser scanning. If a DNA chip was being used, fluorescent-tagged cDNA molecules made from the mRNA present in the infected cell mixture would only bind to complementary sequences on the chip after hybridization, and these could be detected by fluorescence upon laser illumination at the proper wavelength. If DNA-protein interactions were under investigation, laser power could be adjusted to partially atomize some of the protein bound to each spot and its nature could be determined by mass spectroscopy.

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment