Introduction to Genetics

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Chapter 30 Introduction to Genetics

In the broadest sense, genes are simply units of hereditary information; the genome is the totality of all the hereditary information in a cell or organism; and genetics may be defined as the study of genes and genomes. With the advent of modern molecular biology and the Human Genome Project, all aspects of genetics have come to play a more prominent role in the day-to-day evaluation and management of children with neurologic diseases, most of which have a genetic basis. This chapter presents a brief synopsis of the most important principles of genetics, to serve as background for information presented elsewhere in this text. More detailed information on genetics is available in many excellent textbooks, such as Genetics in Medicine [Nussbaum et al., 2007], Genes IX [Lewin, 2007], and Human Molecular Genetics [Strachan and Read, 2010]. Other resources are available from the National Center for Biotechnology Information website (Table 30-1).

Table 30-1 Genetic Information Websites

Site Internet Address
NCBI1 Genetic Disease Websites
GeneTests, GeneReviews2
NCBI1 Genome Data Websites
NCBI1 homepage (Entrez)
dbGaP Genotypes and Phenotypes
dbSNP (SNP database)
Other Genome Data Websites
Ensembl Human Genome Browser
DOE5 Genomics Websites, includes Human Genome Project
UCSC Genome Bioinformatics6

1 National Center for Biotechnology Information.

2 Disease summaries in GeneReviews are authored by experts and peer-reviewed, and so are typically highly accurate and up to date.

3 Disease summaries in OMIM (Online Mendelian Inheritance in Man) are done by staff with oversight and contain both dated and new data; all information from OMIM should be confirmed from a second source.

4 The HUGO Gene Nomenclature Committee website established accepted names for human genes.

5 U.S. Department of Energy Office of Science websites, which include the Human Genome Project website.

6 University of California–Santa Clara Genome Bioinformatics site, which contains the most widely used human genome browser, sometimes called “Golden Path.”

Molecular Basis of Heredity

Modern theories of molecular biology hold that all information needed for function of cells and organisms is contained in macromolecules composed of simple repeating units. The flow of genetic information is (almost) exclusively unidirectional: DNA to RNA to protein. That is, the sequence of deoxyribonucleic acid (DNA) specifies the synthesis and sequence of ribonucleic acid (RNA) by a process known as transcription. Messenger RNA in turn specifies the synthesis and sequence of polypeptides, which are the building blocks of proteins, by a process known as translation. Other forms of RNA function independently. This theory is the central dogma of molecular biology. Accordingly, we begin with a review of the structure and function of these three macromolecules, and continue with reviews of the processes involved in gene and protein expression, including gene structure and organization, RNA processing, and epigenetics. Epigenetics refers to modification of genes other than changes in the DNA sequence, especially by addition of methyl groups to DNA, which alters gene expression. The two most important epigenetic changes found to be relevant to clinical disorders to date are imprinting and X-inactivation.

Structure and Function of DNA

DNA is a large polymer or macromolecule composed of linear sequences of simple repeating units. The specific sequence of these units contains all of the genetic information of an individual cell or organism. The structure of DNA in its native state was deduced by Watson and Crick in 1953 [Watson and Crick, 1953]. The basic repeating unit of DNA is the nucleotide, which consists of a five-carbon sugar known as deoxyribose; a phosphate group; and a nitrogen-containing base, which may be either a purine or a pyrimidine (Figure 30-1A). In DNA, the purine base may be either adenine (A) or guanine (G), and the pyrimidine base may be either thymine (T) or cytosine (C). Nucleotides polymerize into long chains by formation of phosphodiester bonds between the 5′ carbon position of one deoxyribose molecule and the 3′ carbon of the preceding deoxyribose molecule (Figure 30-1B).

Each DNA molecule consists of two strands of nucleotides that are held together by weak hydrogen bonds between pairs of bases: A pairs only with T, and G pairs only with C. These paired units are known as basepairs (bp). In the native state, the two strands wind around each other to form a double helix that resembles a right-hand spiral staircase, with two unequal grooves known as the major and minor grooves (Figure 30-2). A single turn of the helix measures 3.4 nm and contains ten nucleotides. Each strand has a directionality imparted by the deoxyribose sugar backbone. Adjacent nucleotides are linked by phosphodiester bonds between the 5′ and 3′ carbon atoms of the sugar residues, so that one end of the DNA strand has an unlinked 5′ carbon (the 5′ end) and the other end of the strand has an unlinked 3′ carbon atom (the 3′ end). The two strands are antiparallel – that is, they run in opposite directions so that the 5′ end of one strand is paired with the 3′ end of the other. Within living cells, DNA is associated with proteins and supercoiled into more complex structures known as chromosomes, which are described later in the chapter.

Thus, when the sequence of one DNA strand is known, the sequence of the opposite or complementary strand may be predicted. Precise replication of DNA is therefore possible, a process that involves initiation, elongation, and termination stages. The process begins with recognition of an “origin of replication.” Such points of origin are specific DNA sequences, recognized by a protein complex known as the primosome, that occur every 50–300 kilobases (kb) of DNA; the unit kb refers to 1000 sequential nucleotides. The two parental DNA strands must first be separated by helicase, an enzyme that unwinds the supercoiled DNA helix to create a replication fork. The process of elongation occurs at the site of the replication fork or replisome. Synthesis of new strands begins with the addition of approximately ten RNA bases by a protein complex known as primase, and then continues with chain elongation using the original strands as templates. This process is known as semiconservative replication. Both initiation or RNA priming and chain elongation involve large protein complexes that include several DNA polymerases.

Five distinct DNA polymerases have been isolated in mammalian systems, including human cell cultures (Table 30-2). They are able to copy DNA only by adding nucleotides to the 3′ end of the growing chain, so DNA can elongate only in the 5′ to 3′ direction. Thus, the template DNA can be read only in the reverse, or 3′ to 5′, direction. As DNA is unwound, the replication fork necessarily unwinds one strand in the 3′ to 5′ direction and the other in the 5′ to 3′ direction. The 3′ to 5′ or leading strand is replicated in a continuous fashion at the replication fork by DNA polymerases α(I), which primes the reaction, and δ(III), which synthesizes the DNA chain. The new strand is complementary and so elongates in the opposite, or 5′ to 3′, direction.

The 5′ to 3′, or lagging, strand cannot be copied continuously because this would require synthesis of the complementary new strand in a 3′ to 5′ direction, which is not possible, because DNA polymerases are able to synthesize DNA only in the 5′ to 3′ direction. Thus, the lagging strand must be copied by DNA polymerases α(I) and δ(III) in small segments of 100–1000 bp in the opposite direction from the replication fork. These small DNA molecules are known as Okazaki fragments. DNA replication is described as semidiscontinuous because of the continuous replication of the leading strand and the discontinuous replication of the lagging strand. The Okazaki fragments are then joined by another enzyme, DNA ligase. DNA replication is a long process, requiring about 8 hours in most human cells in culture. Thus, the function of DNA is reliably to encode and store the genetic information needed for the cell and organism to function. It has no direct functions itself but rather acts by directing synthesis of both RNA and protein.

Structure and Function of RNA

RNA differs chemically from DNA in the substitution of ribose for deoxyribose in the sugar backbone of the molecule, and of uridine (U) for thymine as one of the pyrimidine bases. Also, RNA normally exists as a single-stranded rather than double-stranded molecule. Recent advances have demonstrated far more diverse functions for RNA than were previously appreciated, particularly involving genes that produce functional RNA products that do not code for proteins. These probably represent at least 5 percent of all human genes, as suggested by current knowledge [Strachan and Read, 2010]. Several distinct classes of RNA molecules have been recognized, most of which are involved with regulating or assisting gene expression.


MicroRNAs (miRNAs) are another class of small noncoding genes that regulate the expression of protein-encoding genes at the post-transcriptional RNA level [Denli et al., 2004]. The process begins with transcription (synthesis) of primary RNA transcripts that range in size from several hundred to several thousand kb. These transcripts are recognized and cut into precursor miRNAs in the nucleus by a protein known as Dicer, moved to the cytoplasm, and processed into mature miRNAs. The mature miRNAs join the RNA-induced silencing complex (RISC), which recognizes and cleaves (or otherwise silences) a target gene. This process has been demonstrated in many organisms, including mammals, and appears likely to play a key role in regulation of many genes.

Structure and Function of Polypeptides and Proteins

Proteins are composed of one or more polypeptide chains. Polypeptides are large polymers or macromolecules composed of linear sequences of repeating units known as amino acids, which are more complex than the repeating units of DNA or RNA. Amino acids consist of a three-carbon backbone, with an amino group attached to carbon 1 and a carboxyl group to carbon 3. They differ in the composition of a side chain attached to carbon 2. With rare exceptions, all polypeptides and proteins in nature are built from different sequences of 20 amino acids (Table 30-3). The side chains may be neutral and hydrophobic, neutral and polar, basic, or acidic. The simplest amino acid is valine, which has a hydrogen ion as the side chain.

Table 30-3 Classification of Amino Acids by Side Chain

Amino Acid 3-letter Code 1-letter Code
Neutral and Hydrophobic
Alanine Ala A
Isoleucine Ile I
Leucine Leu L
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Tryptophan Trp W
Valine Val V
Neutral and Polar
Asparagine Asn N
Cysteine Cys C
Glutamine Glu Q
Glycine Gly G
Serine Ser S
Threonine Thr T
Tyrosine Tyr Y
Aspartic acid Asp D
Glutamic acid Glu E
Arginine Arg R
Histidine His H
Lysine Lys K

The process of information transfer from RNA polypeptides to proteins is known as translation. It relies on the genetic code, the system by which the nucleotide sequence of mRNA specifies the amino acid sequence of a polypeptide chain. In this nearly universal code, each set of three adjacent bases in the mRNA transcript constitutes a codon, and different combinations of bases within the codon specify the individual amino acids (Table 30-4). The small tRNA molecules serve as the molecular link between mRNA codons and amino acids. One segment of each tRNA transcript contains a three-base anticodon that is complementary to a specific codon on the mRNA, whereas another segment contains a binding site for one of the 20 amino acids.

With a total of only 20 amino acids and 64 possible codons, most amino acids are specified by more than one codon. For some of the different amino acids, the base in the third position in the triplet may be either of the purines, either of the pyrimidines, or sometimes any of the four bases. For this reason, the third position in the codon sometimes is called the wobble position. Arginine and leucine are each specified by six codons, whereas only methionine and tryptophan are specified by a single codon. Three codons signal termination of translation and accordingly are called stop codons.


The process of information transfer from RNA to polypeptide or protein is known as translation. This process takes place in the cytoplasm on small structures known as ribosomes, macromolecules composed of the four species of rRNA noted earlier. They function like small migrating factories that travel along an mRNA template, engaging in rapid cycles of peptide bond synthesis. The process consists of initiation, elongation, and termination stages.

The ribosome contains a large site that binds about 35 bp of mRNA, and two adjacent sites for binding the smaller aminoacyl-tRNA molecules. The first is the acceptor or A site, which holds the incoming aminoacyl-tRNA. The second is the donor or P site, which is occupied by a tRNA carrying the growing polypeptide chain. Translation begins with mRNA binding to the ribosome at the site of the first AUG base triplet, which specifies the amino acid methionine, and also serves as the start signal for synthesis of the polypeptide chain and establishes the reading frame of the mRNA.

The mRNA and tRNA then move in the same direction along the ribosome, with the tRNA moving from the “A” site to the “P” site, and the mRNA sliding over three bases, allowing recognition of the next codon. Bonding between the mRNA codon and tRNA anticodon brings the appropriate amino acid into position on the ribosome to form a new peptide bond to the carboxyl end of the growing polypeptide chain. As part of this reaction, the polypeptide chain is released from the tRNA at the “P” site, but remains bonded to the tRNA at the “A” site. The tRNA and mRNA then move another 3 bp along the chain, and the process is repeated. This reaction continues until one of the stop codons is reached. Thus, proteins are synthesized from the amino to the carboxyl terminus, which corresponds to translation from the 5′ to the 3′ end of the mRNA molecule, and methionine is always the first amino acid of each polypeptide chain, although it usually is removed before protein synthesis is completed.

Gene Structure and Organization

As noted earlier, a gene traditionally has been defined as a unit of genetic information. This concept has gradually progressed to a more useful definition, which states that a gene is a sequence of DNA on a chromosome that is required for production of a functional product, which can be either a protein or a functional RNA molecule [Nussbaum et al., 2007]. By convention, genetic information is always read in the 5′ to 3′ direction, whether encoded in DNA or RNA – in an upstream to downstream direction. The nomenclature regarding the 5′ and 3′ positions of the sugar backbone can be confusing. The 5′ carbon of the first nucleotide of a sequence is joined by a phosphodiester bond to a nucleotide not involved in the sequence, whereas its 3′ carbon is joined to the 5′ carbon of the second nucleotide, and so on. The last nucleotide of the sequence has a 3′ carbon, which joins another uninvolved nucleotide.


Genes are composed of a continuous length of DNA with definable start and end points, which include the sequence that codes for the RNA or polypeptide product and is thus known as the coding region. It has become clear, however, that the structure of a gene is complex and includes much more than the coding sequence of the protein. All genes include additional sequences on either end of the coding region – designated the 5′ and 3′ UTRs – that do not code for an RNA product or polypeptide. These regions function to regulate transcription and RNA stability. The gene is considered to include the entire sequence represented in the RNA product because some mutations within noncoding regions can impair gene function.

A model of a typical human gene is shown in Figure 30-3. Promoter sequences required for regulation and initiation of RNA transcription (red diamonds in Figure 30-3) are present at the 5′ end of the gene, such as the CAT and TATA boxes whose sequences are tightly conserved among many different genes and species. Downstream from the promoter sequences is a specific sequence that signals the start of transcription. A short way further downstream is an initiator codon, AUG, which codes for methionine. This triplet is the translation start site, which signals the start of the coding sequence for the polypeptide product. The region between the transcription and translation start sites is the 5′ UTR.

The next segment of the gene is the coding region. The coding regions of most genes in prokaryotes and lower eukaryotes are colinear, which means that the coding sequence corresponds exactly to the sequence of amino acids in the polypeptide. By contrast, most higher eukaryotic genes, including human genes, contain additional sequences that lie within the coding region, interrupting the sequence that represents the polypeptide. The regions that code for the final polypeptide (or functional RNA) product are known as exons, whereas the regions that are missing from the final mRNA product are introns. The removal of introns from the final mRNA product is known as splicing, a complex process that is regulated by a large number of proteins and functional RNA transcripts.

The coding sequence ends at one of three specific stop codons: UAA, UAG, or UGA. The last segment of the gene is the 3′ UTR, which contains a polyadenylation signal and presumably a signal to end transcription, although no transcription stop sequence has been identified. The length of a gene may vary, ranging from less than 1 kb to several hundred kb. The longest gene known, which codes for dystrophin, spans more than 2000 kb of genomic sequence, although this is not the largest protein produced in the cell.

Regulatory Regions

Many genes have highly conserved sequences, a longer distance upstream and downstream of the transcribed gene, that are involved in regulating expression, including enhancers, silencers, locus control regions, and insulators (see Figure 30-3). Enhancer elements function to increase gene expression, while silencers reduce gene expression. Locus control regions may regulate expression of several genes within a chromosome region, while insulators prevent co-regulation of more distant genes and gene regions. All of these are sequences that bind proteins called transcription factors, which can be ubiquitous, tissue-specific, and/or temporally expressed. Promoters are located immediately 5′ of the gene and bind to RNA polymerase II, a necessary step for transcription. Other transcription factors bind upstream of the promoter and activate transcription. Enhancers and silencers are often located at a distance from the promoter, and increase or decrease transcription in a tissue-specific or temporal manner. Overall, the transcription of each gene is tightly regulated, with multiple transcription factors involved.

RNA Processing

Transcription of DNA gives rise to a precursor RNA that corresponds exactly to the genome sequence but must be modified in several ways to become functional, especially for mRNA. The first modification to mRNA is the addition of a CAP structure to the 5′ end and this is followed by the removal or splicing of introns. The mechanism of mRNA splicing depends on the specific nucleotide sequences at the exon/intron boundaries called splice junctions (Figure 30-4). The most important of these is the GT-AG rule: introns almost always start with GT (actually GU, because this occurs in RNA), which is therefore called the splice-donor site, and end with AG, which is called the splice-acceptor site. Several additional specific sequences are also needed, including sequences within the intron just after the GT splice-donor site, at a highly conserved branch site located about 40 bp before the end of the intron and just before the AG splice-acceptor site. The splicing mechanism produces the following:

These reactions are catalyzed by large complexes composed of snRNA and specific proteins. The snRNAs involved have specific sequences that allow binding with conserved intronic sequences or the recognition sites of other snRNAs. The snRNA–protein–target RNA complexes form large particles known as spliceosomes. Once a 5′ splice site is recognized, the complex scans the RNA sequence until it encounters a branch site, which aids in identifying the nearby 3′ splice-acceptor site. This process does not necessarily happen in linear order along the RNA. Rather, the order likely is determined by the vagaries of RNA folding. The last steps involve cleavage of part of the 3′ UTR, which occurs at a specific point downstream from the end of the coding sequence, and addition of a long sequence of adenosine nucleotides that is called the polyA tail. The site of the polyA tail is specified in part by the sequence AAUAAA, which is located within the 3′ UTR.

Imprinting and X-Inactivation

Several regions of the genome are subject to inactivation under special circumstances, with no changes to the DNA sequence. The processes involved thus represent a form of “epigenetic” modification. The two processes reviewed here, imprinting and X-chromosome inactivation, both can result in a phenotype when disrupted.


The process by which certain genes in specific chromosomal regions are expressed from only one chromosome, depending on the parental origin of the chromosome, is known as “imprinting.” Although the mechanism is only partly understood, a key component involves allele-specific DNA methylation, found predominantly at the carbon 5 position of about 80 percent of all cytosines that are part of symmetrical cytosine-guanine (CpG) dinucleotides [Jiang et al., 2004; Strachan and Read, 2010; Weksberg et al., 2003].

This process is controlled by regulatory imprinting “centers,” located nearby on the same chromosome as that of the silenced or “imprinted” gene. In effect, then, two alleles of the same gene that are identical in nucleotide sequence but derived from opposite parents are regulated differently in the same nucleus. This process is reversible, so that the silent, imprinted allele can be reactivated and the active allele silenced when passed through the germline of the opposite-sex parent. Most imprinted genes are found in large clusters of greater than 1 Mb (megabase pairs) in length. Imprinted clusters have been identified in chromosomes 6q24, 7p11.2, 11p15.5, 14q32, 15q11–q13, and 20q13.2, and others may exist as well [Cavaille et al., 2002; Gardner et al., 2000; Hall, 1990; Jiang et al., 2004; Weksberg et al., 2003; Wylie et al., 2000]. Imprinted regions share several common characteristics, including differential DNA methylation, allele-specific RNA transcription, antisense transcripts, histone modifications, and differences in timing of replication.


In mammalian cells with two (or more) X chromosomes, all but one undergo widespread gene silencing by methylation. This phenomenon, known as X-chromosome inactivation (Xi), causes one of the two X chromosomes in cells of female mammals to become transcriptionally inactive early in embryonic development, a phenomenon known as the Lyon hypothesis [Lyon, 1961, 2002]. In mutant cells with more than two X chromosomes, all but one become inactivated. This has the effect of balancing gene dosage of X-linked genes between male and female cells. The process of Xi is random, so that on average the maternally and paternally derived X chromosomes are each inactivated in approximately 50 percent of cells. Changes in this pattern are seen in female carriers of some X-linked diseases, resulting in skewing of Xi. This alteration can be favorable, with decreased severity of the phenotype, or unfavorable, with increased severity of the phenotype [Dobyns et al., 2004].

Cell Cycle and Chromosomal Basis of Heredity

Current knowledge regarding the chromosomal basis of heredity and that concerning the cell cycle are inextricably linked because the intracellular structures now known as chromosomes were first seen in cells undergoing cell division. The existence of chromosomes was foreshadowed by Gregor Mendel’s work. For years after he described independent sorting of genetic traits, occasional exceptions to Mendel’s law of segregation were discovered. Certain traits were found that were typically inherited as a group. These observations were eventually explained by the discovery of chromosomes. The nuclear material of a cell, or chromatin, appears homogeneous during most of the cell cycle, but condenses into distinct rod-shaped organelles during cell division. These tiny structures were called chromosomes because they stain darkly with various biologic dyes.

Cell Cycle

Humans begin life as a single diploid cell or zygote, which gives rise to all of the cells of the body by a combination of cell growth and cell division, with the latter including both asexual (mitosis) and sexual (meiosis) cell division. The life cycle of somatic cells is divided into four stages. After cell division, the cell enters the G1 (gap 1) resting phase, during which DNA synthesis does not occur. Some differentiated cells, such as neurons, stop growth in a modified G1 phase known as G0. Late in G1, the cell passes a critical point, after which it proceeds through the rest of the cell cycle at a standard rate. G1 is followed by the S phase, during which DNA synthesis or replication occurs. The genetic material is duplicated in the form of two chromatids (future chromosomes), joined by attachment to a single centromere. The cell then enters the G2 (gap 2) resting phase, which is much shorter than G1. The G1, S, and G2 phases together constitute interphase.

Chromosomal Basis of Heredity

Chromosome Structure

In humans, the nuclear DNA is dispersed among 46 separate linear structures or chromosomes, each of which consists of a single, uninterrupted double helix that contains 50–250Mb of DNA, and a group of associated proteins that form the support structure or scaffolding. The scaffolding consists of five basic proteins called histones and several more acidic nonhistone proteins. Two copies of each of four histones – H2A, H2B, H3, and H4 – join to form an octamer. The DNA double helix wraps almost twice around the octamer, which involves about 140 bp. Adjacent octamers are separated by a short spacer segment of 20–60bp that is associated with histone H1. The complex of DNA and core histones is known as a nucleosome (see Figure 30-2).

Strings of nucleosomes are further compacted into a secondary helical structure known as a solenoid. These structures have a diameter of about 30 nm (see Figure 30-2) and contain six nucleosomes per turn. The solenoids are packed into large loops of 10–100 kb of DNA, which are attached to a nonhistone protein scaffolding. These loops pack together loosely to form interphase chromosomes. During early prophase, they pack together more closely to form knoblike thickenings known as chromomeres, which then coalesce further to form the bands observed in prometaphase and metaphase chromosomes when stained with appropriate dyes.

The alternating light and dark bands that characterize all nuclear chromosomes with a variety of staining methods likely reflect the compartmentalization of the genome into isochores, defined as large regions with variation in base composition or variable spacing of scaffold attachment regions. The dark bands observed with Giemsa staining are AT-rich, replicate late in the DNA synthesis phase of the cell cycle, and contain relatively few genes. The light bands observed with Giemsa are GC-rich, replicate early, and contain many genes. Some are greatly enriched for GC and contain high concentrations of genes. Most, although not all, such bands are located near the ends or telomeres of chromosomes and therefore are known as T bands.

Specialized Regions

All nuclear chromosomes have specialized regions that are required for chromosome integrity and function, including centromeres, telomeres, and origins of replication. Centromeres are DNA sequences that act in cis. That is, they act on the chromosome on which they are located and are responsible for the segregation of chromosomes during cell division. Centromeres contain extensive repeats of an approximately 171-bp unit known as alpha-satellite DNA, the sequence of which differs slightly between each chromosome. Fragments of chromosomes that lack a centromere, known as acentric fragments, are lost during cell division.

The two ends of a chromosome are called telomeres and also are required for chromosome stability. In humans, they consist of long arrays of tandem repeats of the sequence TTAGGG, which extend about 5–20 kb. DNA polymerases are unable to replicate the telomeres because of the lack of a template. This problem is resolved by the enzyme telomerase, which contains an RNA component to serve as a template to prime further synthesis on the leading strand. Further extension of the leading strand provides the needed template for the lagging strand.

Origins of replication are specialized sequences where DNA replication begins, and thus are important in maintaining chromosome number and integrity. They consist of autonomously replicating sequence elements that contain a core consensus sequence and some imperfect copies with a length of about 50 nucleotides. A consensus human autonomously replicating sequence has been identified [Strachan and Read, 2010].

Regions of variable staining known as heterochromatin consist of long arrays of repeat sequences as short as 5 bp. These regions are located primarily in the pericentromeric regions of chromosomes 1, 9, and 16, and in distal Yq. The five human acrocentric chromosomes have small satellites attached to the short arm by short stalks or secondary constrictions that contain the rRNA genes.

Chromosome Number

Each human somatic cell contains 46 chromosomes that consist of 22 matched pairs known as autosomes and two sex chromosomes: XX in females and XY in males (Figure 30-7). In contrast, human germ cells contain only 23 chromosomes, consisting of 22 unpaired autosomes and a single sex chromosome. The former is known as the diploid or 2n number, and the latter is known as the haploid or 1n number. The autosomes were numbered according to length, with chromosome 1 the longest and chromosome 22 thought to be the shortest. Although chromosome 21 later proved to be shorter than chromosome 22, the numbers were retained for historical reasons. The two members of each pair of autosomes and the two X chromosomes in females carry the same genes and are known as homologous chromosomes, or homologs. Although they appear similar under the microscope, homologs are not strictly identical. They contain the same genes, but the nucleotide sequence differs at thousands of positions.

Organization of the Human Genome

The human genome comprises the total of all genetic information in the cell. It is divided into two separate compartments – a large and complex nuclear genome and a much smaller and simpler mitochondrial genome. The mitochondrial genome consists of a single circular DNA molecule that is present in many copies in each mitochondrion, while the nuclear genome is distributed among the 46 nuclear chromosomes. The available data regarding the genome have become much more extensive and accurate with completion of the Human Genome Project. A few of the most useful Human Genome Project-related websites are listed in Table 30-1.

The Nuclear Genome

The human nuclear genome consists of approximately 3 × 109 bp, or 3000 Mb of DNA. About 75 percent of this represents unique or single-copy DNA, which includes genes and some important regulatory elements. The remaining 25 percent consists of several classes of repetitive DNA [Lander et al., 2001; Nussbaum et al., 2007; Venter et al., 2001].

Genes and Conserved Noncoding DNA

Somewhat surprisingly, recent estimates predict that the human genome contains less than 30,000 protein-coding genes (possibly closer to 20,000) and an uncertain number of other genes producing functional RNA products. This is far fewer than earlier estimates, and accounts for only about 1.2 percent of nuclear DNA [Lander et al., 2001; Venter et al., 2001]. Another 5 percent of the human genome is more conserved than would be expected from estimates of neutral evolution, which suggests that many of these regions have specific, regulatory functions [Chiaromonte et al., 2003; Waterston et al., 2002]. Studies of these highly conserved regions of DNA have used different thresholds, such as stretches of more than 100 bp with 70–80 percent conservation between mouse and human. Some of these regions have been found to contain important noncoding elements [Dermitzakis et al., 2002, 2003; Frazer et al., 2004; Hardison, 2000]. More stringent analysis demonstrates that the human genome contains 481 sequences of 200 or more bp that are 100 percent conserved among human, mouse, and rat [Bejerano et al., 2004]. These segments were designated “ultra-conserved elements,” and are preferentially located near genes involved in RNA processing or regulation of transcription and development. Similarly, about 5000 sequences of 100 bp or more are conserved among these three species, which emphasizes that noncoding sequences are common and important.

Low Copy Repeats

Segmental duplications, also known as low copy repeats (LCRs), are DNA sequences of 10–250 kb, present in multiple copies with greater than 95 percent sequence identity, that make up approximately 5 percent of the human genome [Babcock et al., 2003; Bailey et al., 2002; Cheung et al., 2001; Stankiewicz and Lupski, 2002]. LCRs are dynamic regions of the genome because specific repeats tend to cluster within the same genomic regions, where they mediate unequal nonhomologous recombination events, producing segmental deletions and duplications that are collectively designated “copy number variants” (CNVs). Several of these have been associated with well-known developmental disorders in humans, such as Williams’ syndrome in 7q11.23, Angelman’s syndrome and Prader–Willi syndrome in 15q12, hereditary neuropathy with predisposition to pressure palsies and Charcot–Marie–Tooth neuropathy type 1A in 17p12, Smith–Magenis syndrome in 17p11.2, and DiGeorge’s syndrome in 22q11.2 [Babcock et al., 2003]. Many new CNV-associated devlopmental brain disorders have been described over the past few years.


A mutation is a permanent change in the DNA of an individual organism, specifically a change in the nucleotide sequence anywhere in the genome [Nussbaum et al., 2007]. Genetic diseases and many cancers are caused by mutations that adversely affect function of one or more genes, although most mutations have little or no effect on gene function and therefore do not change the survival or reproductive fitness of an individual. Some of these persist in the population as morphologic variants known as polymorphisms. Sequence changes that have frequencies of less than 1 percent are known as rare variants, whereas those with frequencies of 1 percent or more are known as polymorphisms. By convention, a genetic polymorphism is defined as the occurrence of two or more variants or alleles in a region of DNA where at least two alleles appear with frequencies greater than 1 percent. Several different classes of polymorphisms occur in the genome, and several methods in molecular biology take advantage of the normal variation between individuals.


Microsatellites, also known as satellite DNA or short tandem repeats, are segments of DNA 2–5 nucleotides in length (dinucleotide, trinucleotide, tetranucleotide, or pentanucleotide repeats) that are scattered throughout the genome in noncoding regions between genes or within genes (in introns). They often are used as markers for linkage analysis because of the naturally occurring high variability in repeat number between individuals. These regions are inherently unstable and susceptible to mutations.

The most common microsatellite family consists of 50,000–100,000 cytosine-adenine (CA) repeats, which consist of short tandem repeats of the dinucleotide CA on one strand and guanine-thymine on the complementary strand. They thus take the form (CA)n/(GT)n, with n in the range of 6–30 [Weber and May, 1989]. The number of repeats within a (CA)n block varies greatly among different members of a species, producing a set of alleles that always differ in size by multiples of two bases. About 70 percent of the human population is heterozygous at any given (CA)n repeat locus, making these highly polymorphic. The human genome contains about 50,000–100,000 interspersed (CA)n blocks, which is enough to place 1 block every 30–60 kb, if evenly spaced.

For both VNTR and CA repeat sequences, the combination of high frequency in the genome and a high rate of polymorphism has made them very useful for genetic mapping and association studies. Some microsatellite repeats, most often trinucleotide repeats, present within coding regions of genes or, less often, the 5′ or 3′ UTR, can expand to an abnormal length and are the basis of triplet repeat diseases such as Huntington’s disease, some forms of spinocerebellar ataxia, and fragile X syndrome.

Single-Nucleotide Polymorphisms

Single-nucleotide polymorphisms (SNPs, pronounced “snips”) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is changed. For example, a SNP might change the DNA sequence TCACG to TTACG. The most common sequence change involves replacement of cytosine (C) with thymidine (T), which accounts for about two-thirds of all SNPs. As with other types of sequence variation, a SNP must occur in at least 1 percent of the population to be classified as a polymorphism. SNPs occur in both unique-sequence (coding and noncoding) and repetitive DNA, and are responsible for about 90 percent of human genetic variation. On average, SNPs are found approximately every 100–300 bases along the entire human genome. Although most SNPs likely have no function, some are known to influence disease predisposition or responses to drugs, and thus are proving to be very valuable in studying the causes of common human diseases. The current inventory of known SNPs can be found in the Human SNP database (dbSNP) on the NCBI Entrez website (see Table 30-1).

Restriction enzymes are DNA-cutting enzymes or endonucleases derived from bacteria that cut DNA at specific short sequences found at locations across the entire genome. SNPs can alter the sequences recognized by restriction enzymes, thus adding or removing a cutting site. This is the biological basis for restriction enzyme fragment length polymorphisms (RFLPs). Depending on the location of restriction enzyme sites, specific DNA fragment lengths are obtained on digestion with restriction endonucleases. The presence of a SNP at one of these restriction enzyme sites will affect cleavage and produce two DNA fragments of different sizes that is the RFLP. RFLPs also can be produced by any change that alters the size of the DNA fragment on which the restriction site is located, such as deletions or duplications. RFLPs are a measure of naturally occurring variations or polymorphisms of normal DNA, and are inherited according to mendelian principles. RFLPs have been useful for gene mapping.