Chapter 2

Genomics Basics

It is useful to review the basic concepts of modern molecular biology before fully immersing ourselves in the world of microarrays. We are sure that readers who have had limited exposure to this fast-developing field would appreciate this review, others may skip ahead. Genomics is a fascinating subject; after all, it is the story of life, and can occupy a multivolume book just by itself. In the interest of space, of course, it is necessary that we confine our discussion to those topics that are essential to an understanding of the science underlying microarrays, leaving other topics for interested readers to explore on their own. Some excellent general references that we, not being trained as molecular biologists ourselves, have found useful are listed at the end of the chapter.

2.1 Genes

From ancient times, it was suspected that there existed some sort of a hereditary mechanism that carried information from parent to child. It is because of this mechanism that family members tend to exhibit similar characteristics or traits. For example, they tend to resemble each other in terms of appearance and physical characteristics such as skin color; they tend to be predisposed toward certain diseases such as diabetes, cancer, and heart disease; and so on. However, inheritance is clearly not a perfect copying process. For example, a child of brown-eyed parents could turn out to be blue-eyed. Despite the efforts over the years of many leading scientists and thinkers to understand the hereditary mechanism, its precise nature remained an intriguing mystery until quite recently.

Following centuries of speculation and research, the existence of discrete hereditary units, which we now call genes, has been firmly established. Each gene, either by itself or in combination with some other genes, provides a clear and unambiguous set of instructions for producing some property of its organism. The complete set of genes in an organism, essentially the master blueprint for that organism, is referred to as its genome. This blueprint contains all the hereditary instructions for building, operating, and maintaining the organism, and for passing life in like form on to the next generation of that organism.

Until the twentieth century, there was hardly any concrete information as to what genes were and how they operated. Then, a panoply of innovative research work and pathbreaking discoveries over (roughly) the first half of the twentieth century gave genes a chemical (molecular) existence. This culminated in the pivotal realization that genes are made of deoxyribonucleic acid (DNA).

2.2 Deoxyribonucleic Acid

A DNA molecule consists of two long strands wound tightly around each other in a spiral structure known as a double helix. The structure has been likened to a twisted ladder, whose sides are made of sugar and phosphate and whose rungs are made of bases.

Each strand of the DNA molecule (i.e., each side of the ladder once it has been untwisted and straightened out) is a linear arrangement of repeating similar units called nucleotides. Every nucleotide has three components: a sugar (deoxyribose), a phosphate group, and a nitrogenous base. The base is one of adenine (A), thymine (T), guanine (G), or cytosine (C). The bases on one strand are paired with the bases on the other strand according to the complementary base pairing rules (also called the Watson–Crick base pairing rules): adenine only pairs with thymine, guanine only pairs with cytosine. The pairs so formed are called base pairs (bp); they form the coplanar rungs of the ladder. The force that holds a bp together is a weak hydrogen bond. Even though each individual bond is weak, their cumulative effect along the strands is strong enough to bind the two strands tightly together. As a result, DNA is chemically inert and is a stable carrier of genetic information.

The sequences of bases along each of the two strands of DNA are complementary to each other as they are matched by the complementary base pairing rules. This complementary sequencing has an important consequence. It was recognized from very early on that, whatever the entity was that was a hereditary unit, it must be able to self-replicate so that information could be passed on from generation to generation. At the time that the structure of DNA was deduced, there was a lot of excitement, as it was clear that the complementary structure of the DNA molecule would allow every DNA molecule to create an exact replica of itself, thus fulfilling this requirement.

The DNA replication process is, in principle, quite straightforward. First, the DNA molecule unwinds and the “ladder” unzips, thereby disrupting the weak bonds between the bps and allowing the strands to separate. Then, each strand directs the synthesis of a brand new complementary strand, with free nucleotides matching up with their complementary bases onto each separated strand, a process that produces two descendant DNA molecules. Each descendant consists of one old and one new DNA strand. The constraints imposed by the complementary base pairing rules ensure that each new strand is an exact copy of the old one with the order of the bases along the strands being faithfully preserved.

The preservation of the base order is crucial. The particular order of the bases arranged along any one strand, its DNA sequence, is the mechanism that specifies the exact genetic instructions required to create the traits of a particular organism.

Many genes are located along each long DNA molecule. A gene is a specific contiguous subsequence of the DNA sequence whose A-T-G-C sequence is the code required for constructing a protein. Proteins are giant complex molecules made of chains of amino acids and it is they that are actually both the building blocks and the workhorses of life. Proteins also regulate most of life's day-to-day functions; in fact, even the DNA replication process is mediated by enzymes, proteins whose job is to catalyze biochemical reactions.

2.3 Gene Expression

An organism's DNA is located in its cells. Cells are the fundamental units of all living organisms, both structurally and functionally. A cell is a microscopic, yet extraordinarily complex, structure that contains a heterogeneous mix of substances essential to life.

There are many substructures within a cell. The most prominent one is a highly protected subcompartment called the nucleus, in which resides the organism's DNA. Enclosing the nucleus is the nuclear membrane, the protective wall that separates the nucleus from the rest of the cell, which is called its cytoplasm. The entire cell is enclosed by the plasma membrane. Embedded within this membrane is a variety of protein structures that act as channels and pumps to control the movement of molecules into and out of the cell.

The set of protein-coding instructions in the DNA sequence of a gene resembles a computer program. A computer program must first be compiled and executed in order for anything to happen. In much the same way, a gene must be expressed in order for anything to happen. A gene expresses by transferring its coded information into proteins that dwell in the cytoplasm, a process called gene expression.

The transmission of genetic information from DNA to protein during gene expression is formulated by the central dogma of molecular biology, which can be stated in oversimple terms as “DNA → mRNA → protein.” This postulates that the protein-coding instructions from a gene are transmitted indirectly through messenger RNA (mRNA), a transient intermediary molecule that resembles a single strand of DNA. There are a few differences between mRNA and DNA, three being that mRNA is single stranded, its sugar is ribose, and it has the base uracil (U) rather than the base thymine.

When a gene is expressed, the DNA double helix splits open along its length. One strand of the open helix remains inactive, while the other strand acts as a template against which a complementary strand of mRNA forms (a process called transcription). The sequence of bases along the mRNA strand is identical to the sequence of bases along the inactive DNA strand (except that mRNA has uracil where DNA has thymine). The mRNA strand then separates from the DNA strand and transports out of the nucleus, across the nuclear membrane, and into the cellular cytoplasm. There it serves as the template for protein synthesis, with consecutive (nonoverlapping) triplets of bases (called codons) acting as a code to specify the particular amino acids that make up an individual protein. The sequence of bases along the mRNA is thus converted into a string of amino acids that constitutes the protein molecule for which it codes (a process called translation).

Each possible triplet of mRNA bases codes for a specific amino acid, one of the twenty amino acids that make up proteins. For example, GCC codes for alanine, CAC for histidine, AUC for isoleucine, and GAG for glutamic acid—the complete list is referred to as the genetic code. As there are four possible bases, there are 43=64 possible triplets, but only 20 possible amino acids. This means that there is room for redundancy: for example, GCU, GCC, GCA, and GCG, all code for alanine. This redundancy is a valuable feature of the genetic code as it provides a safeguard against small errors that might occur during transcription.

In addition, the genetic code has specific triplets to signal the start and the end of a...