Introduction

The complete set of instructions for making an organism is called its genome. It contains the master blueprint for all cellular structures and activities for the lifetime of the cell or organism. Found in every nucleus of a persons many trillions of cells, the human genome consists of tightly coiled threads of deoxyribonucleic acid (DNA) and associated protein molecules, organized into structures called chromosomes (Fig. 1: The Human Genome at Four Levels of Detail).

If unwound and tied together, the strands of DNA would stretch more than 5 feet but would be only 50 trillionths of an inch wide. For each organism, the components of these slender threads encode all the information necessary for building and maintaining life, from simple bacteria to remarkably complex human beings. Understanding how DNA performs this function requires some knowledge of its structure and organization.

DNA

In humans, as in other higher organisms, a DNA molecule consists of two strands that wrap around each other to resemble a twisted ladder whose sides, made of sugar and phosphate molecules, are connected by rungs of nitrogen- containing chemicals called bases. Each strand is a linear arrangement of repeating similar units called nucleotides, which are each composed of one sugar, one phosphate, and a nitrogenous base (Fig. 2: DNA Structure). Four different bases are present in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). The particular order of the bases arranged along the sugar- phosphate backbone is called the DNA sequence; the sequence specifies the exact genetic instructions required to create a particular organism with its own unique traits.

The two DNA strands are held together by weak bonds between the bases on each strand, forming base pairs (bp). Genome size is usually stated as the total number of base pairs; the human genome contains roughly 3 billion bp (Fig. 3: Comparison of Largest Known DNA Sequence with Approximate Chromosome and Genome Sizes of Model Organisms and Humans).

Each time a cell divides into two daughter cells, its full genome is duplicated; for humans and other complex organisms, this duplication occurs in the nucleus. During cell division the DNA molecule unwinds and the weak bonds between the base pairs break, allowing the strands to separate. Each strand directs the synthesis of a complementary new strand, with free nucleotides matching up with their complementary bases on each of the separated strands. Strict base- pairing rules are adhered to adenine will pair only with thymine (an A- T pair) and cytosine with guanine (a C- G pair). Each daughter cell receives one old and one new DNA strand (Figs. 1 and 4: DNA Replication). The cells adherence to these base- pairing rules ensures that the new strand is an exact copy of the old one. This minimizes the incidence of errors (mutations) that may greatly affect the resulting organism or its offspring.

Genes

Each DNA molecule contains many genes the basic physical and functional units of heredity. A gene is a specific sequence of nucleotide bases, whose sequences carry the information required for constructing proteins, which provide the structural components of cells and tissues as well as enzymes for essential biochemical reactions. The human genome is estimated to comprise at least 100,000 genes.

Human genes vary widely in length, often extending over thousands of bases, but only about 10% of the genome is known to include the protein- coding sequences (exons) of genes. Interspersed within many genes are intron sequences, which have no coding function. The balance of the genome is thought to consist of other noncoding regions (such as control sequences and intergenic regions), whose functions are obscure. All living organisms are composed largely of proteins; humans can synthesize at least 100,000 different kinds. Proteins are large, complex molecules made up of long chains of subunits called amino acids. Twenty different kinds of amino acids are usually found in proteins. Within the gene, each specific sequence of three DNA bases (codons) directs the cells protein- synthesizing machinery to add specific amino acids. For example, the base sequence ATG codes for the amino acid methionine. Since 3 bases code for 1 amino acid, the protein coded by an average- sized gene (3000 bp) will contain 1000 amino acids. The genetic code is thus a series of codons that specify which amino acids are required to make up specific proteins.

The protein- coding instructions from the genes are transmitted indirectly through messenger ribonucleic acid (mRNA), a transient intermediary molecule similar to a single strand of DNA. For the information within a gene to be expressed, a complementary RNA strand is produced (a process called transcription) from the DNA template in the nucleus. This mRNA is moved from the nucleus to the cellular cytoplasm, where it serves as the template for protein synthesis. The cells protein- synthesizing machinery then translates the codons into a string of amino acids that will constitute the protein molecule for which it codes (Fig. 5: Gene Expression). In the laboratory, the mRNA molecule can be isolated and used as a template to synthesize a complementary DNA (cDNA) strand, which can then be used to locate the corresponding genes on a chromosome map. The utility of this strategy is described in the section on physical mapping.

Chromosomes

The 3 billion bp in the human genome are organized into 24 distinct, physically separate microscopic units called chromosomes. All genes are arranged linearly along the chromosomes. The nucleus of most human cells contains 2 sets of chromosomes, 1 set given by each parent. Each set has 23 single chromosomes22 autosomes and an X or Y sex chromosome. (A normal female will have a pair of X chromosomes; a male will have an X and Y pair.) Chromosomes contain roughly equal parts of protein and DNA; chromosomal DNA contains an average of 150 million bases. DNA molecules are among the largest molecules now known.

Chromosomes can be seen under a light microscope and, when stained with certain dyes, reveal a pattern of light and dark bands reflecting regional variations in the amounts of A and T vs G and C. Differences in size and banding pattern allow the 24 chromosomes to be distinguished from each other, an analysis called a karyotype. A few types of major chromosomal abnormalities, including missing or extra copies of a chromosome or gross breaks and rejoinings (translocations), can be detected by microscopic examination; Downs syndrome, in which an individual's cells contain a third copy of chromosome 21, is diagnosed by karyotype analysis (Fig. 6: Karyotype). Most changes in DNA, however, are too subtle to be detected by this technique and require molecular analysis. These subtle DNA abnormalities (mutations) are responsible for many inherited diseases such as cystic fibrosis and sickle cell anemia or may predispose an individual to cancer, major psychiatric illnesses, and other complex diseases.

Mapping and Sequencing the Human Genome

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