Guide to Analysis of DNA Microarray Data, Second Edition

Book Preface

The fundamental basis of DNA microarrays is the process of hybridization. Two DNA strands hybridize if they are complementary to each other. Complementarity reflects the Watson-Crick rule that adenine (A) binds to thymine (T) and cytosine (C) binds to guanine (G). One or both strands of the DNA hybrid can be replaced by RNA and hybridization will still occur as long as there is complementarity. Hybridization has for decades been used in molecular biology as the basis for such techniques as Southern blotting and Northern blotting. In Southern blotting, a small string of DNA, an oligonucleotide, is used to hybridize to complementary fragments of DNA that have been separated according to size in a gel electrophoresis. If the oligonucleotide is radioactively labeled, the hybridization can be visualized on a photographic film that is sensitive to radiation. In Northern blotting, a radio-labeled oligonucleotide is used to hybridize to messenger RNA that has been run through a gel. If the oligo is specific to a single messenger RNA, then it will bind to the location (band) of that messenger in the gel. The amount of radiation captured on a photographic film depends to some extent on the amount of radio-labeled probe present in the band, which again depends on the amount of messenger. So this method is a semiquantitative detection of individual messengers.

DNA arrays are a massively parallel version of’ Northern and Southern blotting. Instead of distributing the oligonucleotide probes over a gel containing samples of RNA or DNA, the oligonucleotide probes are attached to a surface. Different probes can be attached within micrometers of each other, so it is possible to place many of them on a small surface of one square centimeter, forming a DNA array. The sample is labeled fluorescently and added to the array. After washing away excess unhybridized material, the hybridized material is excited by a laser and is detected by a light scanner that scans the surface of the chip. Because you know the location of each oligonucleotide probe, you can quantify the amount of sample hybridized to it from the image generated by the scan.

There is some contention in the literature on the use of the word “probe” in relation to microarrays. Throughout this book the word “probe” will be used to refer to what is attached to the microarray surface. And the word “target” will be used to refer to what is hybridized to the probes.

Where before it was possible to run a couple of Northern blots or a couple of Southern blots in a day, it is now possible with DNA arrays to run hybridizations for tens of thousands of probes. This has in some sense revolutionized molecular biology and medicine. Instead of studying one gene and one messenger at a time, experimentalists are now studying many genes and many messengers at the same time. In fact, DNA arrays are often used to study all known messengers of an organism. This has opened the possibility of an entirely new, systemic view of how cells react in response to certain stimuli. It is also an entirely new way to study human disease by viewing how it affects the expression of all genes inside the cell. Figure I .2 illustrates the revolution of DNA arrays in biology and medicine by the number of papers published on the topic.