2

DOE Human Genome Program

Primer on Molecular Genetics

Date Published: June 1992

U.S. Department of Energy

Office of Energy Research

Office of Health and Environmental Research

Washington, DC 20585

The "Primer on Molecular Genetics" is taken from the June 1992 DOE Human

Genome 1991-92 Program Report. The primer is intended to be an introduction to

basic principles of molecular genetics pertaining to the genome project.

Human Genome Management Information System

Oak Ridge National Laboratory

1060 Commerce Park

Oak Ridge, TN 37830

Voice: 865/576-6669

Fax: 865/574-9888

E-mail: bkq@ornl.gov
3

Contents

Primer on

Molecular

Genetics

Revised and expanded

by Denise Casey

(HGMIS) from the

primer contributed by

Charles Cantor and

Sylvia Spengler

(Lawrence Berkeley

Laboratory) and

published in the

Human Genome 1989–

90 Program Report.

Introduction ............................................................................................................. 5

DNA............................................................................................................................... 6

Genes............................................................................................................................ 7

Chromosomes ............................................................................................................... 8

Mapping and Sequencing the Human Genome ...................................... 10

Mapping Strategies ..................................................................................................... 11

Genetic Linkage Maps ............................................................................................ 11

Physical Maps......................................................................................................... 13

Low-Resolution Physical Mapping...................................................................... 14

Chromosomal map ......................................................................................... 14

cDNA map ...................................................................................................... 14

High-Resolution Physical Mapping..................................................................... 14

Macrorestriction maps: Top-down mapping ................................................... 16

Contig maps: Bottom-up mapping.................................................................. 17

Sequencing Technologies ........................................................................................... 18

Current Sequencing Technologies ......................................................................... 23

Sequencing Technologies Under Development ..................................................... 24

Partial Sequencing to Facilitate Mapping, Gene Identification ............................... 24

End Games: Completing Maps and Sequences; Finding Specific Genes .................. 25

Model Organism Research.............................................................................. 27

Informatics: Data Collection and Interpretation ..................................... 27

Collecting and Storing Data ........................................................................................ 27

Interpreting Data ......................................................................................................... 28

Mapping Databases .................................................................................................... 29

Sequence Databases .................................................................................................. 29

Nucleic Acids (DNA and RNA)................................................................................ 29

Proteins .................................................................................................................. 30

Impact of the Human Genome Project ....................................................... 30

Glossary... .............................................................................................................. 32
4
5

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

Fig. 1. The Human Genome at Four Levels of Detail. Apart from reproductive cells (gametes) and

mature red blood cells, every cell in the human body contains 23 pairs of chromosomes, each a

packet of compressed and entwined DNA (1, 2). Each strand of DNA consists of repeating

nucleotide units composed of a phosphate group, a sugar (deoxyribose), and a base (guanine,

cytosine, thymine, or adenine) (3). Ordinarily, DNA takes the form of a highly regular doublestranded helix, the strands of which are linked by hydrogen bonds between guanine and cytosine

and between thymine and adenine. Each such linkage is a base pair (bp); some 3 billion bp

constitute the human genome. The specificity of these base-pair linkages underlies the mechanism

of DNA replication illustrated here. Each strand of the double helix serves as a template for the

synthesis of a new strand; the nucleotide sequence (i.e., linear order of bases) of each strand is

strictly determined. Each new double helix is a twin, an exact replica, of its parent. (Figure and

caption text provided by the LBL Human Genome Center.)

the cell or organism. Found in every nucleus of a person’s 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).
6

Primer on

Molecular

Genetics

Deoxyribose


Sugar Molecule

Phosphate Molecule

Nitrogenous


Bases

A T

C G

G C

T A

Weak Bonds


Between


Bases

Sugar-Phosphate


Backbone

Fig. 2. DNA Structure.

The four nitrogenous

bases of DNA are

arranged along the sugarphosphate backbone in a

particular order (the DNA

sequence), encoding all

genetic instructions for an

organism. Adenine (A)

pairs with thymine (T),

while cytosine (C) pairs

with guanine (G). The two

DNA strands are held

together by weak bonds

between the bases.

A gene is a segment of

a DNA molecule (ranging from fewer than

1 thousand bases to

several million), located

in a particular position on

a specific chromosome,

whose base sequence

contains the information

necessary for protein

synthesis.

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). 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).

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 basepairing 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). The

cell’s 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.
7

Fig. 3. Comparison of Largest Known DNA Sequence with Approximate Chromosome and

Genome Sizes of Model Organisms and Humans. A major focus of the Human Genome Project

is the development of sequencing schemes that are faster and more economical.

Largest known continuous DNA sequence

 (yeast chromosome 3)

Escherichia coli (bacterium) genome

Largest yeast chromosome now mapped

Entire yeast genome

Smallest human chromosome (Y)

Largest human chromosome (1)

Entire human genome

350

 4.6

 5.8

 15

 50

250

 3

Comparative Sequence Sizes Bases

Thousand

Million

Million

Million

Million

Million

Billion

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 cell’s 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
8

Primer on

Molecular

Genetics

Fig. 4. DNA Replication.

During replication the DNA

molecule unwinds, with

each single strand

becoming a template for

synthesis of a new,

complementary strand.

Each daughter molecule,

consisting of one old and

one new DNA strand, is an

exact copy of the parent

molecule. [Source:

adapted from Mapping Our

Genes—The Genome

Projects: How Big, How

Fast? U.S. Congress,

Office of Technology

Assessment, OTA-BA-373

(Washington, D.C.: U.S.

Government Printing

Office, 1988).]

G
C


C


A


A
 T


G
C


A
 T


T
 A


C
 G


T
 A


G
C


T
 A


C
 G


A
 T


C
 G


G
C


T


T
 A


C
 G


A
 T


G
 T


A


C


G


A


T


G
 C


A


G


A
 T


A


A
 T


G
C


A


T
 A


C
 G


T
 A


C
 G


G
C


T
 A


C
 G


A
 T


G


G
 C


T


C


G
C


T
 A


C
 G


A
 T


G
C


T
 A


C
 G


A
 T


G
C


T
 A


C
 G


A
 T


ORNL-DWG 91M-17361


T


T


DNA Replication


Parent


Strands


Complementary


New Strand


Complementary


New Strand


mRNA is moved from the nucleus to the cellular cytoplasm, where it serves as the template for protein synthesis. The cell’s 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). 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 chromosomes—22 autosomes and an X or Y sex

chromosome. (A normal female will have a pair of X chromosomes; a male will have an X
9

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;

Down’s syndrome, in which an individual's cells contain a third copy of chromosome 21, is

diagnosed by karyotype analysis (Fig. 6). 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.

Fig. 5. Gene Expression. When genes are expressed, the genetic information (base sequence) on DNA is first transcribed

(copied) to a molecule of messenger RNA in a process similar to DNA replication. The mRNA molecules then leave the cell

nucleus and enter the cytoplasm, where triplets of bases (codons) forming the genetic code specify the particular amino acids that

make up an individual protein. This process, called translation, is accomplished by ribosomes (cellular components composed of

proteins and another class of RNA) that read the genetic code from the mRNA, and transfer RNAs (tRNAs) that transport amino

acids to the ribosomes for attachment to the growing protein. (Source: see Fig. 4.)

NUCLEUS


DNA


Gene
 mRNA


Copying


DNA in


Nucleus


tRNA Bringing


Amino Acid to


Ribosome


Free Amino Acids


Amino


Acids


Growing


Protein Chain


RIBOSOME incorporating


amino acids into the


growing protein chain


CYTOPLASM


ORNL-DWG 91M-17360


mRNA


mRNA

10

Primer on

Molecular

Genetics

Mapping and Sequencing the Human Genome

A primary goal of the Human Genome Project is to make a series of descriptive diagrams—maps—of each human chromosome at increasingly finer resolutions. Mapping

involves (1) dividing the chromosomes into smaller fragments that can be propagated and

char-acterized and (2) ordering (mapping) them to correspond to their respective locations

on the chromosomes. After mapping is completed, the next step is to determine the base

sequence of each of the ordered DNA fragments. The ultimate goal of genome research is

to find all the genes in the DNA sequence and to develop tools for using this information in

the study of human biology and medicine. Improving the instrumentation and techniques

required for mapping and sequencing—a major focus of the genome project—will increase efficiency and cost-effectiveness. Goals include automating methods and optimizing techniques to extract the maximum useful information from maps and sequences.

A genome map describes the order of genes or other markers and the spacing between

them on each chromosome. Human genome maps are constructed on several different

scales or levels of resolution. At the coarsest resolution are genetic linkage maps, which

depict the relative chromosomal locations of DNA markers (genes and other identifiable

DNA sequences) by their patterns of inheritance. Physical maps describe the chemical

characteristics of the DNA molecule itself.

Fig. 6. Karyotype. Microscopic examination of chromosome size and banding patterns allows

medical laboratories to identify and arrange each of the 24 different chromosomes (22 pairs of

autosomes and one pair of sex chromosomes) into a karyotype, which then serves as a tool in the

diagnosis of genetic diseases. The extra copy of chromosome 21 in this karyotype identifies this

individual as having Down’s syndrome.
11

Geneticists have already charted the approximate positions of over 2300 genes, and a

start has been made in establishing high-resolution maps of the genome (Fig. 7). Moreprecise maps are needed to organize systematic sequencing efforts and plan new

research directions.

Mapping Strategies

Genetic Linkage Maps

A genetic linkage map shows the relative locations of specific DNA markers along the

chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. Markers

can be expressed DNA regions (genes) or DNA segments that have no known coding

function but whose inheritance pattern can be followed. DNA sequence differences are

especially useful markers because they are plentiful and easy to characterize precisely.

YEAR


66
 68
 70
 72
 74
 76
 78
 80
 82
 84
 86
 88
 90


0


500


1000


1500


2000


ORNL-DWG 91M-17362A


92


2500


NUMBER OF EXPRESSED GENES MAPPED


Fig. 7. Assignment of Genes

to Specific Chromosomes.

The number of genes assigned

(mapped) to specific chromosomes has greatly increased since

the first autosomal (i.e., not on the

X or Y chromosome) marker was

mapped in 1968. Most of these

genes have been mapped to

specific bands on chromosomes.

The acceleration of chromosome

assignments is due to (1) a combination of improved and new

techniques in chromosome sorting

and band analysis, (2) data from

family studies, and (3) the introduction of recombinant DNA

technology. [Source: adapted from

Victor A. McKusick, “Current

Trends in Mapping Human

Genes,” The FASEB Journal 5(1),

12 (1991).]
12

Primer on

Molecular

Genetics

HUMAN GENOME PROJECT GOALS


Complete a detailed human genetic map


Complete a physical map


Acquire the genome as clones


Determine the complete sequence


Find all the genes


With the data generated by the project, investigators


will determine the functions of the genes and develop


tools for biological and medical applications.


2 Mb


0.1 Mb


5 kb


1 bp


ORNL-DWG 91M-17474


Resolution


HUMAN GENOME PROJECT GOALS

Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist

among individuals so that they are detectable among different members in family studies.

Polymorphisms are variations in DNA sequence that occur on average once every 300 to

500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility. Most variations occur within

introns and have little or no effect on an organism’s appearance or function, yet they are

detectable at the DNA level and can be used as markers. Examples of these types of

markers include (1) restriction fragment length polymorphisms (RFLPs), which reflect

sequence variations in DNA sites that can be cleaved by DNA restriction enzymes (see

box), and (2) variable number of tandem repeat sequences, which are short repeated

sequences that vary in the number of repeated units and, therefore, in length (a characteristic easily measured). The human genetic linkage map is constructed by observing how

frequently two markers are inherited together.

Two markers located near each other on the same chromosome will tend to be passed

together from parent to child. During the normal production of sperm and egg cells, DNA

strands occasionally break and rejoin in different places on the same chromosome or on

the other copy of the same chromosome (i.e., the homologous chromosome). This process

(called meiotic recombination) can result in the separation of two markers originally on the

same chromosome (Fig. 8). The closer the markers are to each other—the more “tightly

linked”—the less likely a recombination event will fall between and separate them. Recombination frequency thus provides an estimate of the distance between two markers.

On the genetic map, distances between markers are measured in terms of centimorgans

(cM), named after the American geneticist Thomas Hunt Morgan. Two markers are said to

be 1 cM apart if they are separated by recombination 1% of the time. A genetic distance of

1 cM is roughly equal to a physical distance of 1 million bp (1 Mb). The current resolution

of most human genetic map regions is about 10 Mb.

The value of the genetic map is that an inherited disease can be located on the map by

following the inheritance of a DNA marker present in affected individuals (but absent in

unaffected individuals), even though the molecular basis of the disease may not yet be

understood nor the responsible gene identified. Genetic maps have been used to find the

exact chromosomal location of several important disease genes, including cystic fibrosis,

sickle cell disease, Tay-Sachs disease, fragile

X syndrome, and myotonic dystrophy.

One short-term goal of the genome project is

to develop a high-resolution genetic map (2 to

5 cM); recent consensus maps of some chromosomes have averaged 7 to 10 cM between

genetic markers. Genetic mapping resolution

has been increased through the application of

recombinant DNA technology, including in vitro

radiation-induced chromosome fragmentation

and cell fusions (joining human cells with those

of other species to form hybrid cells) to create

panels of cells with specific and varied human
13

FATHER
 MOTHER


Marker M


and HD


M


HD


M


HD


M
 M


HD


Marker M


and HD


Marker M


Only*


Marker M


and HD


CHILDREN


*Recombinant: Frequency of this event reflects the distance


 between genes for the marker M and HD.


ORNL-DWG 91M-17363


Fig. 8. Constructing a Genetic

Linkage Map. Genetic linkage

maps of each chromosome are

made by determining how frequently two markers are passed

together from parent to child.

Because genetic material is sometimes exchanged during the production of sperm and egg cells,

groups of traits (or markers) originally together on one chromosome

may not be inherited together.

Closely linked markers are less

likely to be separated by spontaneous chromosome rearrangements. In this diagram, the vertical

lines represent chromosome 4

pairs for each individual in a family.

The father has two traits that can

be detected in any child who

inherits them: a short known DNA

sequence used as a genetic

marker (M) and Huntington’s

disease (HD). The fact that one

child received only a single trait (M)

from that particular chromosome

indicates that the father’s genetic

material recombined during the

process of sperm production. The

frequency of this event helps determine the distance between the two

DNA sequences on a genetic map .

chromosomal components. Assessing the frequency of marker sites remaining together

after radiation-induced DNA fragmentation can establish the order and distance between

the markers. Because only a single copy of a chromosome is required for analysis, even

nonpolymorphic markers are useful in radiation hybrid mapping. [In meiotic mapping

(described above), two copies of a chromosome must be distinguished from each other by

polymorphic markers.]

Physical Maps

Different types of physical maps vary in their degree of resolution. The lowest-resolution

physical map is the chromosomal (sometimes called cytogenetic) map, which is based on

the distinctive banding patterns observed by light microscopy of stained chromosomes. A

cDNA map shows the locations of expressed DNA regions (exons) on the chromosomal

map. The more detailed cosmid contig map depicts the order of overlapping DNA fragments spanning the genome. A macrorestriction map describes the order and distance

between enzyme cutting (cleavage) sites. The highest-resolution physical map is the

complete elucidation of the DNA base-pair sequence of each chromosome in the human

genome. Physical maps are described in greater detail below.
14

Primer on

Molecular

Genetics Low-Resolution Physical Mapping

Chromosomal map. In a chromosomal map, genes or other identifiable DNA fragments

are assigned to their respective chromosomes, with distances measured in base pairs.

These markers can be physically associated with particular bands (identified by cytogenetic staining) primarily by in situ hybridization, a technique that involves tagging the DNA

marker with an observable label (e.g., one that fluoresces or is radioactive). The location

of the labeled probe can be detected after it binds to its complementary DNA strand in an

intact chromosome.

As with genetic linkage mapping, chromosomal mapping can be used to locate genetic

markers defined by traits observable only in whole organisms. Because chromosomal

maps are based on estimates of physical distance, they are considered to be physical

maps. The number of base pairs within a band can only be estimated.

Until recently, even the best chromosomal maps could be used to locate a DNA fragment

only to a region of about 10 Mb, the size of a typical band seen on a chromosome.

Improvements in fluorescence in situ hybridization (FISH) methods allow orientation of

DNA sequences that lie as close as 2 to 5 Mb. Modifications to in situ hybridization

methods, using chromosomes at a stage in cell division (interphase) when they are less

compact, increase map resolution to around 100,000 bp. Further banding refinement

might allow chromosomal bands to be associated with specific amplified DNA fragments,

an improvement that could be useful in analyzing observable physical traits associated

with chromosomal abnormalities.

cDNA map. A cDNA map shows the positions of expressed DNA regions (exons)

relative to particular chromosomal regions or bands. (Expressed DNA regions are those

transcribed into mRNA.) cDNA is synthesized in the laboratory using the mRNA molecule

as a template; base-pairing rules are followed (i.e., an A on the mRNA molecule will pair

with a T on the new DNA strand). This cDNA can then be mapped to genomic regions.

Because they represent expressed genomic regions, cDNAs are thought to identify the

parts of the genome with the most biological and medical significance. A cDNA map can

provide the chromosomal location for genes whose functions are currently unknown. For

disease-gene hunters, the map can also suggest a set of candidate genes to test when

the approximate location of a disease gene has been mapped by genetic linkage techniques.

High-Resolution Physical Mapping

The two current approaches to high-resolution physical mapping are termed “top-down”

(producing a macrorestriction map) and “bottom-up” (resulting in a contig map). With

either strategy (described below) the maps represent ordered sets of DNA fragments that

are generated by cutting genomic DNA with restriction enzymes (see Restriction Enzymes box at right). The fragments are then amplified by cloning or by polymerase chain

reaction (PCR) methods (see DNA Amplification). Electrophoretic techniques are used to

separate the fragments according to size into different bands, which can be visualized by
15

direct DNA staining or by hybridization with DNA probes of interest. The use of purified

chromosomes separated either by flow sorting from human cell lines or in hybrid cell lines

allows a single chromosome to be mapped (see Separating Chromosomes box at right).

A number of strategies can be used to reconstruct the original order of the DNA fragments

in the genome. Many approaches make use of the ability of single strands of DNA and/or

RNA to hybridize—to form double-stranded segments by hydrogen bonding between

complementary bases. The extent of sequence homology between the two strands can be

Separating Chromosomes

Flow sorting

Pioneered at Los Alamos National Laboratory (LANL), flow sorting employs flow

cytometry to separate, according to size, chromosomes isolated from cells during

cell division when they are condensed and stable. As the chromosomes flow singly

past a laser beam, they are differen-tiated by analyzing the amount of DNA present,

and individual chromosomes are directed to specific collection tubes.

Somatic cell hybridization

In somatic cell hybridization, human cells and rodent tumor cells are fused (hybridized); over time, after the chromosomes mix, human chromosomes are preferentially

lost from the hybrid cell until only one or a few remain. Those individual hybrid cells

are then propagated and maintained as cell lines containing specific human chromosomes. Improvements to this technique have generated a number of hybrid cell

lines, each with a specific single human chromosome.

Restriction Enzymes: Microscopic Scalpels

Isolated from various bacteria, restriction enzymes recognize short DNA sequences

and cut the DNA molecules at those specific sites. (A natural biological function of

these enzymes is to protect bacteria by attacking viral and other foreign DNA.) Some

restriction enzymes (rare-cutters) cut the DNA very infrequently, generating a small

number of very large fragments (several thousand to a million bp). Most enzymes cut

DNA more frequently, thus generating a large number of small fragments (less than a

hundred to more than a thousand bp).

On average, restriction enzymes with

• 4-base recognition sites will yield pieces 256 bases long,

• 6-base recognition sites will yield pieces 4000 bases long, and

• 8-base recognition sites will yield pieces 64,000 bases long.

Since hundreds of different restriction enzymes have been characterized, DNA can

be cut into many different small fragments.
16

inferred from the length of the double-stranded segment. Fingerprinting uses restriction

map data to determine which fragments have a specific sequence (fingerprint) in common

and therefore overlap. Another approach uses linking clones as probes for hybridization to

chromosomal DNA cut with the same restriction enzyme.

Macrorestriction maps: Top-down mapping. In top-down mapping, a single

chromosome is cut (with rare-cutter restriction enzymes) into large pieces, which are

ordered and subdivided; the smaller pieces are then mapped further. The resulting macrorestriction maps depict the order of and distance between sites at which rare-cutter

enzymes cleave (Fig. 9a). This approach yields maps with more continuity and fewer gaps

between fragments than contig maps (see below), but map resolution is lower and may

not be useful in finding particular genes; in addition, this strategy generally does not

produce long stretches of mapped sites. Currently, this approach allows DNA pieces to be

located in regions measuring about 100,000 bp to 1 Mb.

The development of pulsed-field gel (PFG) electrophoretic methods has improved the

mapping and cloning of large DNA molecules. While conventional gel electrophoretic

methods separate pieces less than 40 kb (1 kb = 1000 bases) in size, PFG separates

molecules up to 10 Mb, allowing the application of both conventional and new mapping

methods to larger genomic regions.

Primer on

Molecular

Genetics

Fig. 9. Physical Mapping Strategies. Top-down physical mapping (a) produces maps with few gaps, but map resolution may not

allow location of specific genes. Bottom-up strategies (b) generate extremely detailed maps of small areas but leave many gaps.

A combination of both approaches is being used. [Source: Adapted from P. R. Billings et al., “New Techniques for Physical

Mapping of the Human Genome,” The FASEB Journal 5(1), 29 (1991).]

(a)
 (b)


Chromosome
 Linked Library


Detailed but incomplete


Arrayed Library


Fingerprint, map, sequence, or


hybridize to detect overlaps


Macrorestriction Map


Complete but low resolution


Bottom


Up


Top


Down


Contig

17

Contig maps: Bottom-up mapping. The bottom-up approach involves cutting the

chromosome into small pieces, each of which is cloned and ordered. The ordered fragments form contiguous DNA blocks (contigs). Currently, the resulting “library” of clones

varies in size from 10,000 bp to 1 Mb (Fig. 9b). An advantage of this approach is the

accessibility of these stable clones to other researchers. Contig construction can be

verified by FISH, which localizes cosmids to specific regions within chromosomal bands.

Contig maps thus consist of a linked library of small overlapping clones representing a

complete chromosomal segment. While useful for finding genes localized to a small area

(under 2 Mb), contig maps are difficult to extend over large stretches of a chromosome

because all regions are not clonable. DNA probe techniques can be used to fill in the

gaps, but they are time consuming. Figure 10 is a diagram relating the different types of

maps.

Technological improvements now make possible the cloning of large DNA pieces, using

artificially constructed chromosome vectors that carry human DNA fragments as large as

1 Mb. These vectors are maintained in yeast cells as artificial chromosomes (YACs). (For

more explanation, see DNA Amplification.) Before YACs were developed, the largest

cloning vectors (cosmids) carried inserts of only 20 to 40 kb. YAC methodology drastically

reduces the number of clones to be ordered; many YACs span entire human genes. A

more detailed map of a large YAC insert can be produced by subcloning, a process in

which fragments of the original insert are cloned into smaller-insert vectors. Because

some YAC regions are unstable, large-capacity bacterial vectors (i.e., those that can

accommodate large inserts) are also being developed.

GENETIC


MAP


RESTRICTION


FRAGMENTS


ORNL-DWG 91M-17369


ORDERED


LIBRARY


SEQUENCE


Gene or


Polymorphism


Gene or


Polymorphism


Fig. 10. Types of Genome

Maps. At the coarsest resolution,

the genetic map measures

recombination frequency between

linked markers (genes or polymorphisms). At the next resolution level, restriction fragments

of 1 to 2 Mb can be separated

and mapped. Ordered libraries of

cosmids and YACs have insert

sizes from 40 to 400 kb. The base

sequence is the ultimate physical

map. Chromosomal mapping (not

shown) locates genetic sites in

relation to bands on chromosomes (estimated resolution of

5 Mb); new in situ hybridization

techniques can place loci 100 kb

apart. These direct strategies

link the other four mapping

approaches diagramed here.

[Source: see Fig. 9.]
18

Sequencing Technologies

The ultimate physical map of the human genome is the complete DNA sequence—the

determination of all base pairs on each chromosome. The completed map will provide

biologists with a Rosetta stone for studying human biology and enable medical researchers to begin to unravel the mechanisms of inherited diseases. Much effort continues to be

spent locating genes; if the full sequence were known, emphasis could shift to determining

gene function. The Human Genome Project is creating research tools for 21st-century

biology, when the goal will be to understand the sequence and functions of the genes

residing therein.

Achieving the goals of the Human Genome Project will require substantial improvements

in the rate, efficiency, and reliability of standard sequencing procedures. While technological advances are leading to the automation of standard DNA purification, separation, and

detection steps, efforts are also focusing on the development of entirely new sequencing

methods that may eliminate some of these steps. Sequencing procedures currently

involve first subcloning DNA fragments from a cosmid or bacteriophage library into special

sequencing vectors that carry shorter pieces of the original cosmid fragments (Fig. 11).

The next step is to make the subcloned fragments into sets of nested fragments differing

in length by one nucleotide, so that the specific base at the end of each successive

fragment is detectable after the fragments have been separated by gel electrophoresis.

Current sequencing technologies are discussed later.

Primer on

Molecular

Genetics
19

Fig. 11. Constructing Clones for Sequencing. Cloned DNA molecules must be made

progressively smaller and the fragments subcloned into new vectors to obtain fragments small

enough for use with current sequencing technology. Sequencing results are compiled to provide

longer stretches of sequence across a chromosome. (Source: adapted from David A. Micklos and

Greg A. Freyer, DNA Science, A First Course in Recombinant DNA Technology, Burlington, N.C.:

Carolina Biological Supply Company, 1990.)

HUMAN


CHROMOSOME


Average 400,000-bp


fragment cloned into YAC


YEAST ARTIFICIAL CHROMOSOME (YAC)


COSMID


Average 40,000-bp


fragment cloned into cosmid


EcoRI
 EcoRI
 EcoRI
 EcoRI
 EcoRI
 EcoRI
 EcoRI


BamHI
 BamHI
 BamHI
 BamHI
 BamHI
 BamHI
 BamHI


Average 4000-bp


fragment cloned into


plasmid or sequencing


vector
 PLASMID


PARTIAL NUCLEOTIDE SEQUENCE


(from human β-globin gene)


GGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCC


TGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGG. . .


ORNL-DWG 91M-17367


RESTRICTION MAP

20

DNA Amplification:

Cloning and Polymerase

Chain Reaction (PCR)

Cloning (in vivo DNA

amplification)

Cloning involves the use of recombinant DNA

technology to propagate DNA fragments inside a

foreign host. The fragments are usually isolated

from chromosomes using restriction enzymes

and then united with a carrier (a vector). Following introduction into suitable host cells, the DNA

fragments can then be reproduced along with the

host cell DNA. Vectors are DNA molecules

originating from viruses, bacteria, and yeast

cells. They accommodate various sizes of

foreign DNA fragments ranging from 12,000 bp

for bacterial vectors (plasmids and cosmids) to

1 Mb for yeast vectors (yeast artificial chromosomes). Bacteria are most often the hosts for

these inserts, but yeast and mammalian cells

are also used (a).

Cloning procedures provide unlimited material for

experimental study. A random (unordered) set of

cloned DNA fragments is called a library.

Genomic libraries are sets of overlapping fragments encompassing an entire genome (b). Also

available are chromosome-specific libraries,

which consist of fragments derived from source

DNA enriched for a particular chromosome. (See

Separating Chromosomes box.)

Recombinant DNA Molecule

Cut DNA

molecules

with restriction 

enzyme to 

generate 

complementary 

sequences on 

the vector and 

the fragment

Vector DNA

Chromosomal DNA

Fragment

To Be Cloned

Join vector and chromosomal

DNA fragment, using

the enzyme DNA ligase

Introduce into bacterium

Recombinant

DNA Molecule

Bacterial

Chromosome

ORNL-DWG 92M-6649

(a)

(a) Cloning DNA in Plasmids. By fragmenting DNA of any

origin (human, animal, or plant) and inserting it in the DNA of

rapidly reproducing foreign cells, billions of copies of a single

gene or DNA segment can be produced in a very short time.

DNA to be cloned is inserted into a plasmid (a small, selfreplicating circular molecule of DNA) that is separate from

chromosomal DNA. When the recombinant plasmid is introduced into bacteria, the newly inserted segment will be

replicated along with the rest of the plasmid.
21

(b) Constructing an

Overlapping Clone Library.

A collection of clones of

chromosomal DNA, called a

library, has no obvious order

indicating the original positions of the cloned pieces on

the uncut chromosome.

To establish that two particular clones are adjacent to

each other in the genome,

libraries of clones containing

partly overlapping regions

must be constructed. These

clone libraries are ordered by

dividing the inserts into smaller

fragments and determining

which clones share common

DNA sequences.

Restriction Enzyme Cutting Sites

Chromosomal DNA

Partially cut chromosomal DNA with a frequent-cutter 

restriction enzyme (controlling the conditions so that 

not all possible sites are cut on every copy of a specific 

sequence) to generate a series of overlapping fragments 

representing every cutting site in the original sample

Overlapping

Fragments

Cut vector DNA

with a restriction

enzyme Join chromosomal fragments

to vector, using the enzyme

DNA ligase

Library of

Overlapping

Genomic Clones

Chromosomal DNA 

Vector DNA

ORNL-DWG 92M-6650

Vector DNA

(b)
22

PCR (in vitro DNA amplification)

Described as being to genes what Gutenberg’s printing press was to the written word, PCR can amplify a

desired DNA sequence of any origin (virus, bacteria, plant, or human) hundreds of millions of times in a

matter of hours, a task that would have required several days with recombinant technology. PCR is especially valuable because the reaction is highly specific, easily automated, and capable of amplifying minute

amounts of sample. For these reasons, PCR has also had a major impact on clinical medicine, genetic

disease diagnostics, forensic science, and evolutionary biology.

PCR is a process based on a specialized polymerase enzyme, which can synthesize a complementary

strand to a given DNA strand in a mixture containing the 4 DNA bases and 2 DNA fragments (primers, each

about 20 bases long) flanking the target sequence. The mixture is heated to separate the strands of doublestranded DNA containing the target sequence and then cooled to allow (1) the primers to find and bind to

their complementary sequences on the separated strands and (2) the polymerase to extend the primers into

new complementary strands. Repeated heating and cooling cycles multiply the target DNA exponentially,

since each new double strand separates to become two templates for further synthesis. In about 1 hour, 20

PCR cycles can amplify the target by a millionfold.

	TARGET DNA


P1
 P2
 Taq


When heated to 72°C, Taq polymerase extends complementary


strands from primers


First synthesis cycle results


in two copies of


target DNA sequence


DENATURE


DNA


HYBRIDIZE


PRIMERS


EXTEND


NEW DNA


STRANDS


Second synthesis cycle


results in four copies of


target DNA sequence


DNA Amplification Using PCR


FIRST CYCLE
 SECOND CYCLE


Reaction mixture contains target


DNA sequence to be amplified,


two primers (P1, P2), and


heat-stable Taq polymerase


Reaction mixture is heated


tp 95°C to denature target


DNA. Subsequent cooling


to 37°C allows primers to


hybridize to complementary


sequences in target DNA


Source: DNA Science, see Fig. 11.

23

T
 C
 G
 A


G


T


C


G


A


C


T


G


C


A


A


T


2. Sequence read (bottom to top)


 from gel autoradiogram


T
 C
 G
 A


T
 C
 G
 A


1. Sequencing reactions loaded


 onto polyacrylamide gel for


 fragment separation


ORNL-DWG 91M-17368


Current Sequencing Technologies

The two basic sequencing approaches, Maxam-Gilbert and Sanger, differ primarily in the

way the nested DNA fragments are produced. Both methods work because gel electrophoresis produces very high resolution separations of DNA molecules; even fragments

that differ in size by only a single nucleotide can be resolved. Almost all steps in these

sequencing methods are now automated. Maxam-Gilbert sequencing (also called the

chemical degradation method) uses chemicals to cleave DNA at specific bases, resulting

in fragments of different lengths. A refinement to the Maxam-Gilbert method known as

multiplex sequencing enables investigators to analyze about 40 clones on a single DNA

sequencing gel. Sanger sequencing (also called the chain termination or dideoxy method)

involves using an enzymatic procedure to synthesize DNA chains of varying length in four

different reactions, stopping the DNA replication at positions occupied by one of the four

bases, and then determining the resulting fragment lengths (Fig. 12).

These first-generation gel-based sequencing technologies are now being

used to sequence small regions of interest in the human genome. Although

investigators could use existing technology to sequence whole chromosomes, time and cost considerations make large-scale sequencing projects of

this nature impractical. The smallest human chromosome (Y) contains 50 Mb;

the largest (chromosome 1) has 250 Mb. The largest continuous DNA

sequence obtained thus far, however, is approximately 350,000 bp, and the

best available equipment can sequence only 50,000 to 100,000 bases per

year at an approximate cost of $1 to $2 per base. At that rate, an unacceptable 30,000 work-years and at least $3 billion would be required for sequencing alone.

Fig. 12. DNA Sequencing. Dideoxy sequencing (also called chain-termination or

Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying

lengths, stopping DNA replication at one of the four bases and then determining the

resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the

diagram contains

• a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a

new strand of DNA at the point where the primer is hybridized to the template;

• the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend

the DNA strand;

• one labeled deoxynucleotide triphosphate (using a radioactive element or dye); and

• one dideoxynucleotide triphosphate, which terminates the growing chain wherever it

is incorporated. Tube A has didATP, tube C has didCTP, etc.

For example, in the A reaction tube the ratio of the dATP to didATP is adjusted so that

each tube will have a collection of DNA fragments with a didATP incorporated for each

adenine position on the template DNA fragments. The fragments of varying length are

then separated by electrophoresis (1) and the positions of the nucleotides analyzed to

determine sequence. The fragments are separated on the basis of size, with the shorter

fragments moving faster and appearing at the bottom of the gel. Sequence is read from

bottom to top (2). (Source: see Fig. 11.)
24

Sequencing Technologies Under Development

A major focus of the Human Genome Project is the development of automated sequencing technology that can accurately sequence 100,000 or more bases per day at a cost of

less than $.50 per base. Specific goals include the development of sequencing and

detection schemes that are faster and more sensitive, accurate, and economical. Many

novel sequencing technologies are now being explored, and the most promising ones will

eventually be optimized for widespread use.

Second-generation (interim) sequencing technologies will enable speed and accuracy to

increase by an order of magnitude (i.e., 10 times greater) while lowering the cost per base.

Some important disease genes will be sequenced with such technologies as (1) highvoltage capillary and ultrathin electrophoresis to increase fragment separation rate and

(2) use of resonance ionization spectroscopy to detect stable isotope labels.

Third-generation gel-less sequencing technologies, which aim to increase efficiency by

several orders of magnitude, are expected to be used for sequencing most of the human

genome. These developing technologies include (1) enhanced fluorescence detection

of individual labeled bases in flow cytometry, (2) direct reading of the base sequence

on a DNA strand with the use of scanning tunneling or atomic force microscopies,

(3) enhanced mass spectrometric analysis of DNA sequence, and (4) sequencing by

hybridization to short panels of nucleotides of known sequence. Pilot large-scale

sequencing projects will provide opportunities to improve current technologies and will

reveal challenges investigators may encounter in larger-scale efforts.

Partial Sequencing To Facilitate Mapping, Gene

Identification

Correlating mapping data from different laboratories has been a problem because of

differences in generating, isolating, and mapping DNA fragments. A common reference

system designed to meet these challenges uses partially sequenced unique regions (200

to 500 bp) to identify clones, contigs, and long stretches of sequence. Called sequence

tagged sites (STSs), these short sequences have become standard markers for physical

mapping.

Because coding sequences of genes represent most of the potentially useful information

content of the genome (but are only a fraction of the total DNA), some investigators have

begun partial sequencing of cDNAs instead of random genomic DNA. (cDNAs are derived

from mRNA sequences, which are the transcription products of expressed genes.) In addition to providing unique markers, these partial sequences [termed expressed sequence

tags (ESTs)] also identify expressed genes. This strategy can thus provide a means of

rapidly identifying most human genes. Other applications of the EST approach include

determining locations of genes along chromosomes and identifying coding regions in

genomic sequences.

Primer on

Molecular

Genetics
25

End Games: Completing Maps and

Sequences; Finding Specific Genes

Starting maps and sequences is relatively simple; finishing them will require new

strategies or a combination of existing methods. After a sequence is determined using the

methods described above, the task remains to fill in the many large gaps left by current

mapping methods. One approach is single-chromosome microdissection, in which a piece

is physically cut from a chromosomal region of particular interest, broken up into smaller

pieces, and amplified by PCR or cloning (see DNA Amplification). These fragments can

then be mapped and sequenced by the methods previously described.

Chromosome walking, one strategy for filling in gaps, involves hybridizing a primer of

known sequence to a clone from an unordered genomic library and synthesizing a short

complementary strand (called “walking” along a chromosome). The complementary strand

is then sequenced and its end used as the next primer for further walking; in this way the

adjacent, previously unknown, region is identified and sequenced. The chromosome is

thus systematically sequenced from one end to the other. Because primers must be synthesized chemically, a disadvantage of this technique is the large number of different

primers needed to walk a long distance. Chromosome walking is also used to locate

specific genes by sequencing the chromosomal segments between markers that flank the

gene of interest (Fig. 13).

The current human genetic map has about 1000 markers, or 1 marker spaced every

3 million bp; an estimated 100 genes lie between each pair of markers. Higher-resolution

genetic maps have been made in regions of particular interest. New genes can be located

by combining genetic and physical map information for a region. The genetic map basically describes gene order. Rough information about gene location is sometimes available

also, but these data must be used with caution because recombination is not equally likely

at all places on the chromosome. Thus the genetic map, compared to the physical map,

stretches in some places and compresses in others, as though it were drawn on a rubber

band.

The degree of difficulty in finding a disease gene of interest depends largely on what

information is already known about the gene and, especially, on what kind of DNA alterations cause the disease. Spotting the disease gene is very difficult when disease results

from a single altered DNA base; sickle cell anemia is an example of such a case, as are

probably most major human inherited diseases. When disease results from a large DNA

rearrangement, this anomaly can usually be detected as alterations in the physical map of

the region or even by direct microscopic examination of the chromosome. The location of

these alterations pinpoints the site of the gene.

Identifying the gene responsible for a specific disease without a map is analogous to

finding a needle in a haystack. Actually, finding the gene is even more difficult, because

even close up, the gene still looks like just another piece of hay. However, maps give

clues on where to look; the finer the map’s resolution, the fewer pieces of hay to be tested.
26

Once the neighborhood of a gene of interest has been identified, several strategies can be

used to find the gene itself. An ordered library of the gene neighborhood can be constructed if one is not already available. This library provides DNA fragments that can be

screened for additional polymorphisms, improving the genetic map of the region and

further restricting the possible gene location. In addition, DNA fragments from the region

can be used as probes to search for DNA sequences that are expressed (transcribed to

RNA) or conserved among individuals. Most genes will have such sequences. Then

individual gene candidates must be examined. For example, a gene responsible for liver

disease is likely to be expressed in the liver and less likely in other tissues or organs. This

type of evidence can further limit the search. Finally, a suspected gene may need to be

sequenced in both healthy and affected individuals. A consistent pattern of DNA variation

when these two samples are compared will show that the gene of interest has very likely

been found. The ultimate proof is to correct the suspected DNA alteration in a cell and

show that the cell’s behavior reverts to normal.

Primer on

Molecular

Genetics















Probe from


5' flanking


marker is 


used to identify


an overlapping


fragment from a


genomic library


GENOMIC DNA


FRAGMENT


PROBE


Probes from the 3' ends


of cloned fragments are used to


identify successive overlapping


cloned fragments


Chromosome walking continues until a clone is


identified that contains the 3' flanking marker


LINKED FLANKING


MARKER


DISEASE GENE


LINKED FLANKING


MARKER




	ORNL-DWG 91M-17370


5'
 3'




Fig. 13. Cloning a

Disease Gene by

After a marker is linked to

Chromosome Walking. 

within 1 cM of a disease

gene, chromosome

walking can be used to

clone the disease gene

itself. A probe is first

constructed from a

genomic fragment identified from a library as

being the closest linked

marker to the gene. A

restriction fragment

isolated from the end of

the clone near the disease

locus is used to reprobe

the genomic library for an

overlapping clone. This

process is repeated several times to walk across

the chromosome and

reach the flanking marker

on the other side of the

disease-gene locus.

(Source: see Fig. 11.)
27

Model Organism Research

Most mapping and sequencing technologies were developed from studies of nonhuman

genomes, notably those of the bacterium Escherichia coli, the yeast Saccharomyces

cerevisiae, the fruit fly Drosophila melanogaster, the roundworm Caenorhabditis elegans,

and the laboratory mouse Mus musculus. These simpler systems provide excellent

models for developing and testing the procedures needed for studying the much more

complex human genome.

A large amount of genetic information has already been derived from these organisms,

providing valuable data for the analysis of normal gene regulation, genetic diseases, and

evolutionary processes. Physical maps have been completed for E. coli, and extensive

overlapping clone sets are available for S. cerevisiae and C. elegans. In addition,

sequencing projects have been initiated by the NIH genome program for E. coli,

S. cerevisiae, and C. elegans.

Mouse genome research will provide much significant comparative information because of

the many biological and genetic similarities between mouse and man. Comparisons of

human and mouse DNA sequences will reveal areas that have been conserved during

evolution and are therefore important. An extensive database of mouse DNA sequences

will allow counterparts of particular human genes to be identified in the mouse and extensively studied. Conversely, information on genes first found to be important in the mouse

will lead to associated human studies. The mouse genetic map, based on morphological

markers, has already led to many insights into human biology. Mouse models are being

developed to explore the effects of mutations causing human diseases, including diabetes, muscular dystrophy, and several cancers. A genetic map based on DNA markers is

presently being constructed, and a physical map is planned to allow direct comparison

with the human physical map.

Informatics: Data Collection and Interpretation

Collecting and Storing Data

The reference map and sequence generated by genome

research will be used as a primary information source for

human biology and medicine far into the future. The vast

amount of data produced will first need to be collected,

stored, and distributed. If compiled in books, the data

would fill an estimated 200 volumes the size of a Manhattan telephone book (at 1000 pages each), and reading it

would require 26 years working around the clock (Fig.14).

Because handling this amount of data will require extensive use of computers, database development will be a

major focus of the Human Genome Project. The present

challenge is to improve database design, software for

HUMAN GENETIC DIVERSITY:


The Ultimate Human Genetic Database

Any two individuals differ in about 3 x 106 bases (0.1%).


The population is now about 5 x 109. 


A catalog of all sequence differences would require


15 x 1015 entries.


This catalog may be needed to find the rarest or most


complex disease genes.
28

database access and manipulation, and data-entry procedures to compensate for the

varied computer procedures and systems used in different laboratories. Databases need

to be designed that will accurately represent map information (linkage, STSs, physical

location, disease loci) and sequences (genomic, cDNAs, proteins) and link them to each

other and to bibliographic text databases of the scientific and medical literature.

Interpreting Data

New tools will also be needed for analyzing the data from genome maps and sequences.

Recognizing where genes begin and end and identifying their exons, introns, and regulatory sequences may require extensive comparisons with sequences from related species

such as the mouse to search for conserved similarities (homologies). Searching a database for a particular DNA sequence may uncover these homologous sequences in a

known gene from a model organism, revealing insights into the function of the corresponding human gene.

Correlating sequence information with genetic linkage data and disease gene research

will reveal the molecular basis for human variation. If a newly identified gene is found to

code for a flawed protein, the altered protein must be compared with the normal version

to identify the specific abnormality that causes disease. Once the error is pinpointed,

researchers must try to determine how to correct it in the human body, a task that will

require knowledge about how the protein functions and in which cells it is active.

Fig. 14. Magnitude of

Genome Data. If the DNA

sequence of the human

genome were compiled in

books, the equivalent of

200 volumes the size of a

Manhattan telephone book

(at 1000 pages each)

would be needed to hold

it all. New data-analysis

tools will be needed

for understanding the

information from genome

maps and sequences.

Primer on

Molecular

Genetics

HUMAN GENOME
 200 Telephone Books


(1000 pages each)


Drosophila (fruit fly)


yeast


E. coli (bacterium)


yeast chromosome 3


10 books


 1 book


300 pages


 14 pages


(longest continuous sequence now known)


ORNL-DWG 91M-17472


Model Organism Genomes

29

Correct protein function depends on the three-dimensional

(3D), or folded, structure the proteins assume in biological

environments; thus, understanding protein structure will be

essential in determining gene function. DNA sequences

will be translated into amino acid sequences, and researchers will try to make inferences about functions either

by com-paring protein sequences with each other or by

comparing their specific 3-D structures (Fig. 15).

Because the 3-D structure patterns (motifs) that protein

molecules assume are much more evolutionarily conserved than amino acid sequences, this type of homology

search could prove more fruitful. Particular motifs may

serve similar functions in several different proteins, information that would be valuable in genome analyses.

Currently, however, only a few protein motifs can be recognized at the sequence level.

Continued development of analytic capabilities to facilitate grouping protein sequences

into motif families will make homology searches more successful.

Mapping Databases

The Genome Data Base (GDB), located at Johns Hopkins University (Baltimore, Maryland), provides location, ordering, and distance information for human genetic markers,

probes, and contigs linked to known human genetic disease. GDB is presently working on

incorporating physical mapping data. Also at Hopkins is the Online Mendelian Inheritance

in Man database, a catalog of inherited human traits and diseases.

The Human and Mouse Probes and Libraries Database (located at the American Type

Culture Collection in Rockville, Maryland) and the GBASE mouse database (located at

Jackson Laboratory, Bar Harbor, Maine) include data on RFLPs, chromosomal assignments, and probes from the laboratory mouse.

Sequence Databases

Nucleic Acids (DNA and RNA)

Public databases containing the complete nucleotide sequence of the human genome and

those of selected model organisms will be one of the most useful products of the Human

Genome Project. Four major public databases now store nucleotide sequences: GenBank

and the Genome Sequence DataBase (GSDB) in the United States, European Molecular

Biology Laboratory (EMBL) Nucleotide Sequence Database in the United Kingdom, and

the DNA Database of Japan (DDBJ). The databases collaborate to share sequences,

which are compiled from direct author submissions and journal scans. The four databases

now house a total of almost 200 Mb of sequence. Although human sequences predominate, more than 8000 species are represented. [Paragraph updated July 1994]

Fig. 15. Understanding

Gene Function.

Understanding how

genes function will

require analyses of the

3-D structures of the

proteins for which the

genes code.

GENE
 PROTEIN


FUNCTION
 STRUCTURE


 ORNL-DWG 91M-17473

30

Proteins

The major protein sequence databases are the Protein Identification Resource (National

Biomedical Research Foundation), Swissprot, and GenPept (both distributed with

GenBank). In addition to sequence information, they contain information on protein motifs

and other features of protein structure.

Impact of the Human Genome Project

The atlas of the human genome will revolutionize medical practice and biological

research into the 21st century and beyond. All human genes will eventually be found, and

accurate diagnostics will be developed for most inherited diseases. In addition, animal

models for human disease research will be more easily developed, facilitating the understanding of gene function in health and disease.

Researchers have already identified single genes associated with a number of diseases,

such as cystic fibrosis, Duchenne muscular dystrophy, myotonic dystrophy, neurofibromatosis, and retinoblastoma. As research progresses, investigators will also uncover the

mechanisms for diseases caused by several genes or by a gene interacting with environmental factors. Genetic susceptibilities have been implicated in many major disabling and

fatal diseases including heart disease, stroke, diabetes, and several kinds of cancer. The

identification of these genes and their proteins will pave the way to more-effective

therapies and preventive measures. Investigators determining the underlying biology of

genome organization and gene regulation will also begin to understand how humans

develop from single cells to adults, why this process sometimes goes awry, and what

changes take place as people age.

New technologies developed for genome research will also find myriad applications in

industry, as well as in projects to map (and ultimately improve) the genomes of economically important farm animals and crops.

While human genome research itself does not pose any new ethical dilemmas, the use of

data arising from these studies presents challenges that need to be addressed before the

data accumulate significantly. To assist in policy development, the ethics component of

the Human Genome Project is funding conferences and research projects to identify and

consider relevant issues, as well as activities to promote public awareness of these topics.

Primer on

Molecular

Genetics
31
32

Portions of the

glossary text were

taken directly or

modified from definitions in the U.S.

Congress Office of

Technology Assessment document:

Mapping Our

Genes—The Genome

Projects: How Big,

How Fast? OTA-BA373, Washington,

D.C.: U.S. Government Printing Office,

April 1988.

Adenine (A): A nitrogenous base, one member of the base pair A-T (adenine-thymine).

Alleles: Alternative forms of a genetic locus; a single allele for each locus is inherited

separately from each parent (e.g., at a locus for eye color the allele might result in blue or

brown eyes).

Amino acid: Any of a class of 20 molecules that are combined to form proteins in living

things. The sequence of amino acids in a protein and hence protein function are determined by the genetic code.

Amplification: An increase in the number of copies of a specific DNA fragment; can be in

vivo or in vitro. See cloning, polymerase chain reaction.

Arrayed library: Individual primary recombinant clones (hosted in phage, cosmid, YAC,

or other vector) that are placed in two-dimensional arrays in microtiter dishes. Each

primary clone can be identified by the identity of the plate and the clone location (row and

column) on that plate. Arrayed libraries of clones can be used for many applications,

including screening for a specific gene or genomic region of interest as well as for physical

mapping. Information gathered on individual clones from various genetic linkage and

physical map analyses is entered into a relational database and used to construct physical

and genetic linkage maps simultaneously; clone identifiers serve to interrelate the multilevel maps. Compare library, genomic library.

Autoradiography: A technique that uses X-ray film to visualize radioactively labeled

molecules or fragments of molecules; used in analyzing length and number of DNA

fragments after they are separated by gel electrophoresis.

Autosome: A chromosome not involved in sex determination. The diploid human genome

consists of 46 chromosomes, 22 pairs of autosomes, and 1 pair of sex chromosomes (the

X and Y chromosomes).

Bacteriophage: See phage.

Base pair (bp): Two nitrogenous bases (adenine and thymine or guanine and cytosine)

held together by weak bonds. Two strands of DNA are held together in the shape of a

double helix by the bonds between base pairs.

Base sequence: The order of nucleotide bases in a DNA molecule.

Base sequence analysis: A method, sometimes automated, for determining the base

sequence.

Biotechnology: A set of biological techniques developed through basic research and now

applied to research and product development. In particular, the use by industry of recombinant DNA, cell fusion, and new bioprocessing techniques.

bp: See base pair.

cDNA: See complementary DNA.

Glossary
33

Centimorgan (cM): A unit of measure of recombination frequency. One centimorgan is

equal to a 1% chance that a marker at one genetic locus will be separated from a marker

at a second locus due to crossing over in a single generation. In human beings, 1 centimorgan is equivalent, on average, to 1 million base pairs.

Centromere: A specialized chromosome region to which spindle fibers attach during cell

division.

Chromosomes: The self-replicating genetic structures of cells containing the cellular

DNA that bears in its nucleotide sequence the linear array of genes. In prokaryotes,

chromosomal DNA is circular, and the entire genome is carried on one chromosome.

Eukaryotic genomes consist of a number of chromosomes whose DNA is associated with

different kinds of proteins.

Clone bank: See genomic library.

Clones: A group of cells derived from a single ancestor.

Cloning: The process of asexually producing a group of cells (clones), all genetically

identical, from a single ancestor. In recombinant DNA technology, the use of DNA manipulation procedures to produce multiple copies of a single gene or segment of DNA is

referred to as cloning DNA.

Cloning vector: DNA molecule originating from a virus, a plasmid, or the cell of a higher

organism into which another DNA fragment of appropriate size can be integrated without

loss of the vector’s capacity for self-replication; vectors introduce foreign DNA into host

cells, where it can be reproduced in large quantities. Examples are plasmids, cosmids,

and yeast artificial chromosomes; vectors are often recombinant molecules containing

DNA sequences from several sources.

cM: See centimorgan.

Code: See genetic code.

Codon: See genetic code.

Complementary DNA (cDNA): DNA that is synthesized from a messenger RNA template; the single-stranded form is often used as a probe in physical mapping.

Complementary sequences: Nucleic acid base sequences that can form a doublestranded structure by matching base pairs; the complementary sequence to G-T-A-C is

C-A-T-G.

Conserved sequence: A base sequence in a DNA molecule (or an amino acid sequence

in a protein) that has remained essentially unchanged throughout evolution.

Contig map: A map depicting the relative order of a linked library of small overlapping

clones representing a complete chromosomal segment.
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Contigs: Groups of clones representing overlapping regions of a genome.

Cosmid: Artificially constructed cloning vector containing the cos gene of phage lambda.

Cosmids can be packaged in lambda phage particles for infection into E. coli; this permits

cloning of larger DNA fragments (up to 45 kb) than can be introduced into bacterial hosts

in plasmid vectors.

Crossing over: The breaking during meiosis of one maternal and one paternal chromosome, the exchange of corresponding sections of DNA, and the rejoining of the chromosomes. This process can result in an exchange of alleles between chromosomes. Compare recombination.

Cytosine (C): A nitrogenous base, one member of the base pair G-C (guanine and

cytosine).

Deoxyribonucleotide: See nucleotide.

Diploid: A full set of genetic material, consisting of paired chromosomes—one chromosome from each parental set. Most animal cells except the gametes have a diploid set of

chromosomes. The diploid human genome has 46 chromosomes. Compare haploid.

DNA (deoxyribonucleic acid): The molecule that encodes genetic information. DNA is a

double-stranded molecule held together by weak bonds between base pairs of nucleotides. The four nucleotides in DNA contain the bases: adenine (A), guanine (G), cytosine

(C), and thymine (T). In nature, base pairs form only between A and T and between G and

C; thus the base sequence of each single strand can be deduced from that of its partner.

DNA probes: See probe.

DNA replication: The use of existing DNA as a template for the synthesis of new DNA

strands. In humans and other eukaryotes, replication occurs in the cell nucleus.

DNA sequence: The relative order of base pairs, whether in a fragment of DNA, a gene,

a chromosome, or an entire genome. See base sequence analysis.

Domain: A discrete portion of a protein with its own function. The combination of domains

in a single protein determines its overall function.

Double helix: The shape that two linear strands of DNA assume when bonded together.

E. coli: Common bacterium that has been studied intensively by geneticists because of its

small genome size, normal lack of pathogenicity, and ease of growth in the laboratory.

Electrophoresis: A method of separating large molecules (such as DNA fragments or

proteins) from a mixture of similar molecules. An electric current is passed through a

medium containing the mixture, and each kind of molecule travels through the medium at

a different rate, depending on its electrical charge and size. Separation is based on these

differences. Agarose and acrylamide gels are the media commonly used for electrophoresis of proteins and nucleic acids.

Glossary
35

Endonuclease: An enzyme that cleaves its nucleic acid substrate at internal sites in the

nucleotide sequence.

Enzyme: A protein that acts as a catalyst, speeding the rate at which a biochemical

reaction proceeds but not altering the direction or nature of the reaction.

EST: Expressed sequence tag. See sequence tagged site.

Eukaryote: Cell or organism with membrane-bound, structurally discrete nucleus and

other well-developed subcellular compartments. Eukaryotes include all organisms except

viruses, bacteria, and blue-green algae. Compare prokaryote. See chromosomes.

Evolutionarily conserved: See conserved sequence.

Exogenous DNA: DNA originating outside an organism.

Exons: The protein-coding DNA sequences of a gene. Compare introns.

Exonuclease: An enzyme that cleaves nucleotides sequentially from free ends of a linear

nucleic acid substrate.

Expressed gene: See gene expression.

FISH (fluorescence in situ hybridization): A physical mapping approach that uses

fluorescein tags to detect hybridization of probes with metaphase chromosomes and with

the less-condensed somatic interphase chromatin.

Flow cytometry: Analysis of biological material by detection of the light-absorbing or

fluorescing properties of cells or subcellular fractions (i.e., chromosomes) passing in a

narrow stream through a laser beam. An absorbance or fluorescence profile of the sample

is produced. Automated sorting devices, used to fractionate samples, sort successive

droplets of the analyzed stream into different fractions depending on the fluorescence

emitted by each droplet.

Flow karyotyping: Use of flow cytometry to analyze and/or separate chromosomes on

the basis of their DNA content.

Gamete: Mature male or female reproductive cell (sperm or ovum) with a haploid set of

chromosomes (23 for humans).

Gene: The fundamental physical and functional unit of heredity. A gene is an ordered

sequence of nucleotides located in a particular position on a particular chromosome that

encodes a specific functional product (i.e., a protein or RNA molecule). See gene expression.

Gene expression: The process by which a gene’s coded information is converted into the

structures present and operating in the cell. Expressed genes include those that are

transcribed into mRNA and then translated into protein and those that are transcribed into

RNA but not translated into protein (e.g., transfer and ribosomal RNAs).
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Glossary

Gene families: Groups of closely related genes that make similar products.

Gene library: See genomic library.

Gene mapping: Determination of the relative positions of genes on a DNA molecule

(chromosome or plasmid) and of the distance, in linkage units or physical units, between

them.

Gene product: The biochemical material, either RNA or protein, resulting from expression

of a gene. The amount of gene product is used to measure how active a gene is; abnormal amounts can be correlated with disease-causing alleles.

Genetic code: The sequence of nucleotides, coded in triplets (codons) along the mRNA,

that determines the sequence of amino acids in protein synthesis. The DNA sequence of

a gene can be used to predict the mRNA sequence, and the genetic code can in turn be

used to predict the amino acid sequence.

Genetic engineering technologies: See recombinant DNA technologies.

Genetic map: See linkage map.

Genetic material: See genome.

Genetics: The study of the patterns of inheritance of specific traits.

Genome: All the genetic material in the chromosomes of a particular organism; its size is

generally given as its total number of base pairs.

Genome projects: Research and technology development efforts aimed at mapping and

sequencing some or all of the genome of human beings and other organisms.

Genomic library: A collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. Compare library,

arrayed library.

Guanine (G): A nitrogenous base, one member of the base pair G-C (guanine and

cytosine).

Haploid: A single set of chromosomes (half the full set of genetic material), present in the

egg and sperm cells of animals and in the egg and pollen cells of plants. Human beings

have 23 chromosomes in their reproductive cells. Compare diploid.

Heterozygosity: The presence of different alleles at one or more loci on homologous

chromosomes.

Homeobox: A short stretch of nucleotides whose base sequence is virtually identical in

all the genes that contain it. It has been found in many organisms from fruit flies to human

beings. In the fruit fly, a homeobox appears to determine when particular groups of genes

are expressed during development.
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Homologies: Similarities in DNA or protein sequences between individuals of the same

species or among different species.

Homologous chromosomes: A pair of chromosomes containing the same linear gene

sequences, each derived from one parent.

Human gene therapy: Insertion of normal DNA directly into cells to correct a genetic

defect.

Human Genome Initiative: Collective name for several projects begun in 1986 by DOE

to (1) create an ordered set of DNA segments from known chromosomal locations, (2)

develop new computational methods for analyzing genetic map and DNA sequence data,

and (3) develop new techniques and instruments for detecting and analyzing DNA. This

DOE initiative is now known as the Human Genome Program. The national effort, led by

DOE and NIH, is known as the Human Genome Project.

Hybridization: The process of joining two complementary strands of DNA or one each of

DNA and RNA to form a double-stranded molecule.

Informatics: The study of the application of computer and statistical techniques to the

management of information. In genome projects, informatics includes the development of

methods to search databases quickly, to analyze DNA sequence information, and to

predict protein sequence and structure from DNA sequence data.

In situ hybridization: Use of a DNA or RNA probe to detect the presence of the complementary DNA sequence in cloned bacterial or cultured eukaryotic cells.

Interphase: The period in the cell cycle when DNA is replicated in the nucleus; followed

by mitosis.

Introns: The DNA base sequences interrupting the protein-coding sequences of a gene;

these sequences are transcribed into RNA but are cut out of the message before it is

translated into protein. Compare exons.

In vitro: Outside a living organism.

Karyotype: A photomicrograph of an individual’s chromosomes arranged in a standard

format showing the number, size, and shape of each chromosome type; used in lowresolution physical mapping to correlate gross chromosomal abnormalities with the

characteristics of specific diseases.

kb: See kilobase.

Kilobase (kb): Unit of length for DNA fragments equal to 1000 nucleotides.

Library: An unordered collection of clones (i.e., cloned DNA from a particular organism),

whose relationship to each other can be established by physical mapping. Compare

genomic library, arrayed library.
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Linkage: The proximity of two or more markers (e.g., genes, RFLP markers) on a chromosome; the closer together the markers are, the lower the probability that they will be

separated during DNA repair or replication processes (binary fission in prokaryotes,

mitosis or meiosis in eukaryotes), and hence the greater the probability that they will be

inherited together.

Linkage map: A map of the relative positions of genetic loci on a chromosome, determined on the basis of how often the loci are inherited together. Distance is measured in

centimorgans (cM).

Localize: Determination of the original position (locus) of a gene or other marker on a

chromosome.

Locus (pl. loci): The position on a chromosome of a gene or other chromosome marker;

also, the DNA at that position. The use of locus is sometimes restricted to mean regions

of DNA that are expressed. See gene expression.

Macrorestriction map: Map depicting the order of and distance between sites at which

restriction enzymes cleave chromosomes.

Mapping: See gene mapping, linkage map, physical map.

Marker: An identifiable physical location on a chromosome (e.g., restriction enzyme

cutting site, gene) whose inheritance can be monitored. Markers can be expressed

regions of DNA (genes) or some segment of DNA with no known coding function but

whose pattern of inheritance can be determined. See RFLP, restriction fragment length

polymorphism.

Mb: See megabase.

Megabase (Mb): Unit of length for DNA fragments equal to 1 million nucleotides and

roughly equal to 1 cM.

Meiosis: The process of two consecutive cell divisions in the diploid progenitors of sex

cells. Meiosis results in four rather than two daughter cells, each with a haploid set of

chromosomes.

Messenger RNA (mRNA): RNA that serves as a template for protein synthesis. See

genetic code.

Metaphase: A stage in mitosis or meiosis during which the chromosomes are aligned

along the equatorial plane of the cell.

Mitosis: The process of nuclear division in cells that produces daughter cells that are

genetically identical to each other and to the parent cell.

mRNA: See messenger RNA.

Multifactorial or multigenic disorders: See polygenic disorders.

Glossary
39

Multiplexing: A sequencing approach that uses several pooled samples simultaneously,

greatly increasing sequencing speed.

Mutation: Any heritable change in DNA sequence. Compare polymorphism.

Nitrogenous base: A nitrogen-containing molecule having the chemical properties of a

base.

Nucleic acid: A large molecule composed of nucleotide subunits.

Nucleotide: A subunit of DNA or RNA consisting of a nitrogenous base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a phosphate molecule, and a sugar molecule (deoxyribose in DNA and ribose in RNA). Thousands of nucleotides are linked to form a DNA or RNA molecule. See DNA, base pair,

RNA.

Nucleus: The cellular organelle in eukaryotes that contains the genetic material.

Oncogene: A gene, one or more forms of which is associated with cancer. Many

oncogenes are involved, directly or indirectly, in controlling the rate of cell growth.

Overlapping clones: See genomic library.

PCR: See polymerase chain reaction.

Phage: A virus for which the natural host is a bacterial cell.

Physical map: A map of the locations of identifiable landmarks on DNA (e.g., restriction

enzyme cutting sites, genes), regardless of inheritance. Distance is measured in base

pairs. For the human genome, the lowest-resolution physical map is the banding patterns

on the 24 different chromosomes; the highest-resolution map would be the complete

nucleotide sequence of the chromosomes.

Plasmid: Autonomously replicating, extrachromosomal circular DNA molecules, distinct

from the normal bacterial genome and nonessential for cell survival under nonselective

conditions. Some plasmids are capable of integrating into the host genome. A number of

artificially constructed plasmids are used as cloning vectors.

Polygenic disorders: Genetic disorders resulting from the combined action of alleles of

more than one gene (e.g., heart disease, diabetes, and some cancers). Although such

disorders are inherited, they depend on the simultaneous presence of several alleles; thus

the hereditary patterns are usually more complex than those of single-gene disorders.

Compare single-gene disorders.

Polymerase chain reaction (PCR): A method for amplifying a DNA base sequence using

a heat-stable polymerase and two 20-base primers, one complementary to the (+)-strand

at one end of the sequence to be amplified and the other complementary to the (–)-strand

at the other end. Because the newly synthesized DNA strands can subsequently serve

as additional templates for the same primer sequences, successive rounds of primer
40

annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined

sequence in a DNA sample.

Polymerase, DNA or RNA: Enzymes that catalyze the synthesis of nucleic acids on

preexisting nucleic acid templates, assembling RNA from ribonucleotides or DNA from

deoxyribonucleotides.

Polymorphism: Difference in DNA sequence among individuals. Genetic variations

occurring in more than 1% of a population would be considered useful polymorphisms for

genetic linkage analysis. Compare mutation.

Primer: Short preexisting polynucleotide chain to which new deoxyribonucleotides can be

added by DNA polymerase.

Probe: Single-stranded DNA or RNA molecules of specific base sequence, labeled

either radioactively or immunologically, that are used to detect the complementary base

sequence by hybridization.

Prokaryote: Cell or organism lacking a membrane-bound, structurally discrete nucleus

and other subcellular compartments. Bacteria are prokaryotes. Compare eukaryote. See

chromosomes.

Promoter: A site on DNA to which RNA polymerase will bind and initiate transcription.

Protein: A large molecule composed of one or more chains of amino acids in a specific

order; the order is determined by the base sequence of nucleotides in the gene coding for

the protein. Proteins are required for the structure, function, and regulation of the body’s

cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, and antibodies.

Purine: A nitrogen-containing, single-ring, basic compound that occurs in nucleic acids.

The purines in DNA and RNA are adenine and guanine.

Pyrimidine: A nitrogen-containing, double-ring, basic compound that occurs in nucleic

acids. The pyrimidines in DNA are cytosine and thymine; in RNA, cytosine and uracil.

Rare-cutter enzyme: See restriction enzyme cutting site.

Recombinant clones: Clones containing recombinant DNA molecules. See recombinant

DNA technologies.

Recombinant DNA molecules: A combination of DNA molecules of different origin that

are joined using recombinant DNA technologies.

Glossary
41

Recombinant DNA technologies: Procedures used to join together DNA segments in a

cell-free system (an environment outside a cell or organism). Under appropriate conditions, a recombinant DNA molecule can enter a cell and replicate there, either autonomously or after it has become integrated into a cellular chromosome.

Recombination: The process by which progeny derive a combination of genes different

from that of either parent. In higher organisms, this can occur by crossing over.

Regulatory regions or sequences: A DNA base sequence that controls gene expression.

Resolution: Degree of molecular detail on a physical map of DNA, ranging from low to

high.

Restriction enzyme, endonuclease: A protein that recognizes specific, short nucleotide

sequences and cuts DNA at those sites. Bacteria contain over 400 such enzymes that

recognize and cut over 100 different DNA sequences. See restriction enzyme cutting site.

Restriction enzyme cutting site: A specific nucleotide sequence of DNA at which a

particular restriction enzyme cuts the DNA. Some sites occur frequently in DNA (e.g.,

every several hundred base pairs), others much less frequently (rare-cutter; e.g., every

10,000 base pairs).

Restriction fragment length polymorphism (RFLP): Variation between individuals in

DNA fragment sizes cut by specific restriction enzymes; polymorphic sequences that

result in RFLPs are used as markers on both physical maps and genetic linkage maps.

RFLPs are usually caused by mutation at a cutting site. See marker.

RFLP: See restriction fragment length polymorphism.

Ribonucleic acid (RNA): A chemical found in the nucleus and cytoplasm of cells; it plays

an important role in protein synthesis and other chemical activities of the cell. The structure of RNA is similar to that of DNA. There are several classes of RNA molecules,

including messenger RNA, transfer RNA, ribosomal RNA, and other small RNAs, each

serving a different purpose.

Ribonucleotides: See nucleotide.

Ribosomal RNA (rRNA): A class of RNA found in the ribosomes of cells.

Ribosomes: Small cellular components composed of specialized ribosomal RNA and

protein; site of protein synthesis. See ribonucleic acid (RNA).

RNA: See ribonucleic acid.

Sequence: See base sequence.
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Sequence tagged site (STS): Short (200 to 500 base pairs) DNA sequence that has a

single occurrence in the human genome and whose location and base sequence are

known. Detectable by polymerase chain reaction, STSs are useful for localizing and

orienting the mapping and sequence data reported from many different laboratories and

serve as landmarks on the developing physical map of the human genome. Expressed

sequence tags (ESTs) are STSs derived from cDNAs.

Sequencing: Determination of the order of nucleotides (base sequences) in a DNA or

RNA molecule or the order of amino acids in a protein.

Sex chromosomes: The X and Y chromosomes in human beings that determine the sex

of an individual. Females have two X chromosomes in diploid cells; males have an X and

a Y chromosome. The sex chromosomes comprise the 23rd chromosome pair in a

karyotype. Compare autosome.

Shotgun method: Cloning of DNA fragments randomly generated from a genome. See

library, genomic library.

Single-gene disorder: Hereditary disorder caused by a mutant allele of a single gene

(e.g., Duchenne muscular dystrophy, retinoblastoma, sickle cell disease). Compare

polygenic disorders.

Somatic cells: Any cell in the body except gametes and their precursors.

Southern blotting: Transfer by absorption of DNA fragments separated in electrophoretic

gels to membrane filters for detection of specific base sequences by radiolabeled complementary probes.

STS: See sequence tagged site.

Tandem repeat sequences: Multiple copies of the same base sequence on a chromosome; used as a marker in physical mapping.

Technology transfer: The process of converting scientific findings from research laboratories into useful products by the commercial sector.

Telomere: The ends of chromosomes. These specialized structures are involved in the

replication and stability of linear DNA molecules. See DNA replication.

Thymine (T): A nitrogenous base, one member of the base pair A-T (adenine-thymine).

Transcription: The synthesis of an RNA copy from a sequence of DNA (a gene); the first

step in gene expression. Compare translation.

Transfer RNA (tRNA): A class of RNA having structures with triplet nucleotide sequences

that are complementary to the triplet nucleotide coding sequences of mRNA. The role of

tRNAs in protein synthesis is to bond with amino acids and transfer them to the ribosomes, where proteins are assembled according to the genetic code carried by mRNA.

Glossary
43

Transformation: A process by which the genetic material carried by an individual cell is

altered by incorporation of exogenous DNA into its genome.

Translation: The process in which the genetic code carried by mRNA directs the synthesis

of proteins from amino acids. Compare transcription.

tRNA: See transfer RNA.

Uracil: A nitrogenous base normally found in RNA but not DNA; uracil is capable of

forming a base pair with adenine.

Vector: See cloning vector.

Virus: A noncellular biological entity that can reproduce only within a host cell. Viruses

consist of nucleic acid covered by protein; some animal viruses are also surrounded by

membrane. Inside the infected cell, the virus uses the synthetic capability of the host to

produce progeny virus.

VLSI: Very large-scale integration allowing over 100,000 transistors on a chip.

YAC: See yeast artificial chromosome.

Yeast artificial chromosome (YAC): A vector used to clone DNA fragments (up to 400

kb); it is constructed from the telomeric, centromeric, and replication origin sequences

needed for replication in yeast cells. Compare cloning vector, cosmid.
44