Dairy Biotechnology

Module 3. Genetic engineering technology / recombinant DNA technology

Lesson 15
DNA MICROARRAY

15.1 Introduction

DNA microarray is a genomic tool used in molecular biology to monitor the expression of thousands of genes simultaneously on a single chip, thus providing the opportunity to analyse the enormous genetic data in one experiment. The developments are occurring daily making this technology the central platform for functional genomics. DNA microarrays can be used to analyse and measure gene expression, detect single nucleotide polymorphism (SNP), genotype or resequence mutant genomes, comparative genome analysis, drug discovery and biodiversity etc. Their high throughput capabilities also hold tremendous potential for pathogen detection, identification, and genotyping in molecular diagnostic laboratories.

15.2 Definition

Microarray is defined as an array of orderly arranged probes consisting of specific single stranded DNA sequences either short oligonucleotides or cDNAs arrayed on chips (also known as biochips, DNA array, gene chip etc.) which can be used to hybridize with a cDNA sample under high stringency conditions of hybridization.

15.3 Principle of Microarray

The basic principle of DNA microarray is based on complementary base pairing. The DNA double helix’s complementary base pairing specificity is exploited in DNA microarray. They are composed of ‘DNA probes’ that are spotted onto a solid substrate i.e glass, silicon etc. Each spot on the support consists of probes that are complementary to the target gene or gene of interest. The basis behind this is hybridization. In hybridization, two single stranded DNA join together to form double stranded DNA due to their complementarity. Hybridized targets can then be detected using one of the several available reporter systems.

15.4 DNA Microarray Technique

DNA microarray technique involves the following basic steps.

i) Preparation of probes
ii) Construction of DNA array
iii) Preparation of samples
iv) Hybridization
v) Detection / Scanner / Reader

15.4.1 Preparation of probes

The probes to be spotted on to the array include single stranded short oligonucleotides ranging in length from 9-50 mer. For genotypic microarrays, probes could be the PCR products from the cloned genes.

15.4.2 Construction of DNA array

The probes are printed onto microscopic slides robotically in a specific grid pattern. The delivery of samples on chip can also be performed manually. The delivery technologies used extensively for construction of arrays are:

15.4.2.1 Mechanical microspotting

The use of prefabricated oligonucleotides is the simplest mode to construct microarrays. The oligonucleotide probes can be deposited straight from a reagent tray onto the chemically modified glass surface by a printhead containing microspotting pins, tweezers, capillaries or micropipettes. After the first spotting cycle, the pin is washed and reloaded to deposit on an adjacent spot. This process is repeated. The steps are speeded up by using robotic control.

15.4.2.2 ‘Ink jet’ or ‘Piezoelectric printing’

Minute volume of reagents are delivered to defined locations on the slide. The printhead moves across the array and at each spot electrical stimulation causes the DNA bases, cDNAs or other molecules to be delivered onto the surface via tiny jets.

15.4.3 Preparation of samples

Samples constitute messenger RNA (mRNA) of the cells or tissues of interest. The RNA thus obtained is converted to cDNA (complementary DNA) using reverse transcriptase. During this reaction, cDNA is labeled with fluorescent dyes like Cy3 (red), Cy5 (green), or radioactive nucleotides. The two different samples are labeled with two different colored dyes. The labeled cDNAs are then hybridized to DNA arrays. These steps have also been shown in Fig. 15.1.

Fig. 15.1 Steps in a microarray experiment

15.4.4 Hybridization

The labeled cDNA is then hybridized to the microarray slide. Hybridization is the reaction that occurs between the labeled fluorescent cDNA sample with probes on the glass slide. If the target cDNA has complementarity to the probe, it will bind to these probes. The hybridization conditions vary with the type of application of array as some require low stringency (low temperature and high salt) while others require high stringency (high temperature and low salt).

15.4.5 Detection / Scanner / Reader

After hybridization, the microarray is scanned using scanners either based on epifluorescent or laser confocal scanning microscope, which illuminates each spot of DNA and separately measures the fluorescence for each dye or CCD based detection systems. CCD detection systems can even detect very small quantities of array bound molecules. The data thus obtained can be used to find the relative abundance of the sequences of each specific gene in the messenger RNA or DNA samples. The hybridization pattern can then be used to identify the genes that are expressed differentially in the tissues or cells.

15.5 Microarray Equipment

15.5.1 Arrayers

Arrayers are available from several companies e.g. Affymetrix, Agilent, Molecular dynamics, Synteni, Genome systems etc. but are highly expensive. With improvement of technology, these may become cheaper in near future. An arrayer produced by Biorobotics (Micro GridII) is shown in Fig. 15.2.

Fig. 15.2 DNA arrayers – microGrid II (Biorobotics)

15.5.2 Scanners

The principle of scanners is to detect the different levels of fluorescence between the spots on the microarray. The basic principle is that the light source inside the scanner excites the fluorescently labeled samples which is then detected, measured and recorded. The scanners from Affymetrix and Molecular Dynamics is also shown Fig. 15.3.

Fig. 15.3 DNA scanners from affymetrix and molecular dynamics

15.6 Applications

Microarrays have a number of applications in different areas that may further expand in future.

1. DNA sequencing by hybridization
2. Single nucleotide polymorphism and point mutations
3. Functional Genomics
4. Diagnostics and Genetic Mapping
5. Reverse genetics and DNA chips
6. Genomic mismatch scanning
7. Disease diagnostics
8. Proteomics
9. Transcriptomics

Books

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

Introduction to Biotechnology, Brown, C.M., Campbell, I and Priest, F.G. Panima Publishing Corporation, 2005. ISBN : 81-86535-42-X

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2^nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Molecular Biotechnology : Principles and Applications of Recombinant DNA, Glick, B.R., Pasternack, Jack, J and Patten, Cheryl, L (Eds)., 4^th Washington., ASM Press, 2010. ISBN : 1-55581-498-4

Molecular Biology and Biotechnology : a guide for students, Krauzer, H. And Massey, A.(Eds) 3^rd Washington DC : ASM Press, 2008, ISBN : 978-155581-4724

Recombinant DNA and Biotechnology : a guide for teachers, Kreuzer, H and Massey, A. 2^nd Washington : ASM Press, 2001, ISBN : 155581-175-2

Molecular Biotechnology, Primrose, S.B. 2^nd New Delhi : Panima, 2001. ISBN : 81-86535-21-7

Molecular Biology and Biotechnology, Smith, C.A. and Wood, E.J. London : Chapman and Hall, 1991.ISBN : 0-412-40750-7

Introduction to Biotechnology, Thieman, W.J and Pallidino, M.A. 2nd New York : Pearson, 2009, ISBN : 978-0-321-58903-3

Molecular Biology and Biotechnology, Walker, J.M and Rapley, R. 4^th – New Delhi : Panima Publishing Corporation, 2003, ISBN : 81-86535-40-3

Gene Biotechnology, Wu William, Welsh, M.J., Kaufman, P.B and Zhang, H.H. 2^nd Boca Raton : CRC press, 2004. ISBN : 0-8493-1288-4

From Genes to Genomes: Concepts and Applications of DNA Technology, 3rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6

Microbial Genetics, 2^nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974

14. Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Internet Resources

http://en.wikipedia.org/wiki/DNA_microarray

www.youtube.com/watch?v=VNsThMNjKhM

www.sciencedaily.com/articles/d/dna_microarray.htm

http://www.genome.wisc.edu/functional/microarray.htm

http://en.wikipedia.org/wiki/DNA_microarray_experiment