GENERAL BIOLOGY PRE-FINAL REVIEWER The tight pairing of the homologous chromosomes is

called synapsis.
Sexual reproduction requires fertilization: the union of two
cells from two individual organisms. The synaptonemal complex also supports the exchange
Haploid cells contain one set of chromosomes. Cells of chromosomal segments between non-sister
containing two sets of chromosomes are called diploid. homologous chromatids in a process called crossing
over. The crossover events are the first source of
The number of sets of chromosomes in a cell is called its genetic variation produced by meiosis.
ploidy level.
At the end of prophase I, the pairs are held together
Sexual reproduction is the production of haploid cells only at the chiasmata; they are called tetrads because
(gametes) and the fusion (fertilization) of two gametes to form the four sister chromatids of each pair of homologous
a single, unique diploid cell called a zygote.
chromosomes are now visible.
Haploid cells that are part of the sexual reproductive cycle are
Prometaphase I
produced by a type of cell division called meiosis.
Prometaphase I is the formation of the spindle fiber
Meiosis I is the first round of meiotic division and consists
of prophase I, prometaphase I, and so on. Meiosis II, the apparatus where spindle fiber microtubules attach to
second round of meiotic division, includes prophase II, the kinetochore proteins at the centromeres.
prometaphase II, and so on At the end of prometaphase I, each tetrad is attached to
microtubules from both poles, with one homologous
chromosome facing each pole. In addition, the nuclear
The Process of Meiosis - Meiosis I membrane has broken down entirely.
Meiosis I
Metaphase I
Meiosis is preceded by an interphase consisting of three
During metaphase I, the tetrads move to the
stages. The G1 phase (also called the first gap phase)
metaphase plate with kinetochores facing opposite
initiates this stage and is focused on cell growth. The S
poles.
phase is next, during which the DNA of the
chromosomes is replicated. This replication produces There are two possibilities for orientation at the
two identical copies, called sister chromatids, that are metaphase plate. The possible number of alignments,
held together at the centromere by cohesin proteins. therefore, equals 2n, where n is the number of
The centrosomes, which are the structures that chromosomes per set. Given these two mechanisms, it
organize the microtubules of the meiotic spindle, also is highly unlikely that any two haploid cells resulting
replicate. Finally, during the G2 phase (also called the from meiosis will have the same genetic composition.
second gap phase), the cell undergoes the final
Anaphase I
preparations for meiosis.
In anaphase I, the microtubules pull the attached
chromosomes apart. The sister chromatids remain
Prophase I tightly bound together at the centromere.
During prophase I, chromosomes condense and Telophase I and Cytokinesis
become visible inside the nucleus. As the nuclear
In telophase I, the separated chromosomes arrive at
envelope begins to break down, homologous
opposite poles. In some organisms, the chromosomes
chromosomes move closer together.
decondense and nuclear envelopes form around the
Synaptonemal complex- a lattice of proteins between chromatids in telophase I. Then cytokinesis, the
the homologous chromosomes, forms at specific physical separation of the cytoplasmic components
locations, spreading to cover the entire length of the into two daughter cells, occurs without reformation of
chromosomes. the nuclei.
Two haploid cells are the end result of the first meiotic The Process of Meiosis - Comparing Meiosis and
division. The cells are haploid because at each pole Mitosis
there is just one of each pair of the homologous
Mitosis and meiosis are both forms of division of the
chromosomes. Therefore, only one full set of the
nucleus in eukaryotic cells.
chromosomes is present. Although there is only one
chromosome set, each homolog still consists of two The purpose of mitosis is cell regeneration, growth, and
sister chromatids. asexual reproduction, while the purpose of meiosis is
the production of gametes for sexual reproduction.
Mitosis is a single nuclear division that results in two
The Process of Meiosis - Meiosis II nuclei that are usually partitioned into two new
daughter cells.
Meiosis II
In contrast, meiosis consists of two nuclear divisions
Meiosis II initiates immediately after cytokinesis,
resulting in four nuclei that are usually partitioned into
usually before the chromosomes have fully
four new haploid daughter cells.
decondensed. In contrast to meiosis I, meiosis II
resembles a normal mitosis. The main differences between mitosis and meiosis
occur in meiosis I. In meiosis I, the homologous
During meiosis II, the sister chromatids within the two
chromosome pairs become associated with each other
daughter cells separate, forming four new haploid
and are bound together with the synaptonemal
gametes. The mechanics of meiosis II is similar to
complex.
mitosis, except that each dividing cell has only one set
of homologous chromosomes. Meiosis I is referred to as a reduction division. There is
no such reduction in ploidy level during mitosis.
Prophase II
Meiosis II is not a reduction division because, although
If the chromosomes decondensed in telophase I, they
there are fewer copies of the genome in the resulting
condense again. If nuclear envelopes were formed, they
cells, there is still one set of chromosomes, as there was
fragment into vesicles.
at the end of meiosis I. Meiosis II is, therefore, referred
Prometaphase II to as equatorial division.

The nuclear envelopes are completely broken down and

the spindle is fully formed. Each sister chromatid forms
Sexual Reproduction - Advantages and Disadvantages
an individual kinetochore that attaches to microtubules
of Sexual Reproduction
from opposite poles.
Sexual reproduction was an early evolutionary
Metaphase II
innovation after the appearance of eukaryotic cells.
The sister chromatids are maximally condensed and During sexual reproduction, the genetic material of two
aligned at the equator of the cell. individuals is combined to produce genetically-diverse
offspring that differ from their parents.
Anaphase II
The process of meiosis produces unique reproductive
The sister chromatids are pulled apart by the cells called gametes, which have half the number of
kinetochore microtubules and move toward opposite chromosomes as the parent cell. Fertilization, the
poles. Non-kinetochore microtubules elongate the cell. fusion of haploid gametes from two individuals,
Telophase II and Cytokinesis restores the diploid condition.

The chromosomes arrive at opposite poles and begin to There are three main categories of sexual life cycles:
decondense. Nuclear envelopes form around the diploid-dominant, demonstrated by most animals;
chromosomes. Cytokinesis separates the two cells into haploid-dominant, demonstrated by all fungi and some
four unique haploid cells. At this point, the newly- algae; and the alternation of generations,
formed nuclei are both haploid. demonstrated by plants and some algae.
Sexual reproduction: Sexual reproduction is the Haploid-Dominant Life Cycle
creation of a new organism by combining the genetic
Within haploid-dominant life cycles, the multicellular
material of two organisms. There are two main
haploid stage is the most obvious life stage. Most fungi
processes during sexual reproduction: meiosis, involving
and algae employ a life cycle type in which the “body”
the halving of the number of chromosomes, and
of the organism, the ecologically important part of the
fertilization, involving the fusion of two gametes and the
life cycle, is haploid.
restoration of the original number of chromosomes.
Alternation of Generations
Asexual reproduction: any form of reproduction that
involves neither meiosis nor fusion of gametes The third life-cycle type, employed by some algae and
all plants, is a blend of the haploid-dominant and
The Red Queen Hypothesis
diploid-dominant extremes. Species with alternation of
Red Queen hypothesis, first proposed by Leigh Van generations have both haploid and diploid multicellular
Valen in 1973. organisms as part of their life cycle. The haploid
multicellular plants are called gametophytes because
The Red Queen’s catchphrase was, “It takes all the
they produce gametes from specialized cells.
running you can do to stay in the same place.” This is
an apt description of co-evolution between competing In plants such as moss, the gametophyte organism is
species. the free-living plant, while the sporophyte is physically
dependent on the gametophyte. In other plants, such
as ferns, both the gametophyte and sporophyte plants
Sexual Reproduction - Life Cycles of Sexually are free-living; however, the sporophyte is much larger.
Reproducing Organisms
zygote: a diploid fertilized egg cell
In sexual reproduction, the genetic material of two
gametophyte: a plant (or the haploid phase in its life
individuals is combined to produce genetically diverse
cycle) that produces gametes by mitosis in order to
offspring that differ from their parents. Fertilization
produce a zygote
and meiosis alternate in sexual life cycles.
sporophyte: a plant (or the diploid phase in its life cycle)
There are three main categories of life cycles in
that produces spores by meiosis in order to produce
eukaryotic organisms: diploid-dominant, haploid-
gametophytes
dominant, and alternation of generations.

Diploid-Dominant Life Cycle

Introduction to Mendelian Inheritance
In the diploid-dominant life cycle, the multicellular
diploid stage is the most obvious life stage, as occurs Genetics is the study of heredity, or the passing of
with most animals, including humans. Nearly all animals traits from parents to offspring. Gregor Johann Mendel
employ a diploid-dominant life cycle strategy in which set the framework for genetics long before
the only haploid cells produced by the organism are the chromosomes or genes had been identified, at a time
gametes. when meiosis was not well understood. For his work,
Mendel is often referred to as the “father of modern
Early in the development of the embryo, specialized
genetics.”
diploid cells, called germ cells, are produced within the
gonads (e.g. testes and ovaries). Germ cells are capable Mendel grew and studied around 29,000 garden pea
of mitosis to perpetuate the cell line and meiosis to plants in a monastery’s garden, where he analyzed
produce gametes. seven characteristics of the garden pea plants: flower
color (purple or white), seed texture (wrinkled or
Fertilization occurs with the fusion of two gametes,
round), seed color (yellow or green), stem length (long
usually from different individuals, restoring the diploid
or short), pod color (yellow or green), pod texture
state.
(inflated or constricted), and flower position (axial or
terminal). Based on the appearance, or phenotypes, of
the seven traits, Mendel developed genotypes for those then collected and grew the seeds from the F1 plants to
traits. produce the F2, or second filial, generation. Mendel’s
experiments extended beyond the F2 generation to the
Because of Mendel’s work, the fundamental principles
F3 and F4generations, and so on, but it was the ratio of
of heredity were revealed, which are often referred to
characteristics in the P0−F1−F2 generations that were
as Mendel’s Laws of Inheritance. We now know that
the most intriguing and became the basis for Mendel’s
genes, carried on chromosomes, are the basic functional
postulates.
units of heredity with the capability to be replicated,
expressed, or mutated.

Mendel’s Model System Rules of Probability for Mendelian Inheritance

Mendel’s seminal work was accomplished using the Key Points

garden pea, Pisum sativum, to study inheritance. Pea
The Product Rule is used to determine the outcome of
plant reproduction is easily manipulated; large
an event with two independent events; the probability
quantities of garden peas could be cultivated
of the event is the product of the probabilities of each
simultaneously, allowing Mendel to conclude that his
individual event.
results did not occur simply by chance.
The Sum Rule is used to determine the outcome of an
phenotype: the observable characteristics of an
event with two mutually exclusive events from
organism, often resulting from its genetic information or
multiple pathways; the probability of the event is the
a combination of genetic information and
sum of the probabilities of each individual event.
environmental factors
The Product Rule of probability is used to determine
genotype: the specific genetic information of a cell or
the probability of having both dominant traits in the
organism, usually a description of the allele or alleles
F2progeny; it is the product of the probabilities of
relating to a specific gene.
having the dominant trait for each characteristic.
true-breeding plant: a plant that always produces
The Sum Rule of probability is used to determine the
offspring of the same phenotype when self-fertilized;
probability of having one dominant trait in the F2
one that is homozygous for the trait being followed.
generation of a dihybrid cross; it is the sum of the
Mendelian Crosses probabilities of each individual with that trait.

Mendel performed crosses, which involved mating two

true-breeding individuals that have different traits. In
Chromosomal Theory of Inheritance
the pea, which is a naturally self-pollinating plant, this
is done by manually transferring pollen from the anther In 1902, Theodor Boveri observed that proper
of a mature pea plant of one variety to the stigma of a embryonic development of sea urchins does not occur
separate mature pea plant of the second variety. In unless chromosomes are present. That same year,
plants, pollen carries the male gametes (sperm) to the Walter Sutton observed the separation of
stigma, a sticky organ that traps pollen and allows the chromosomes into daughter cells during meiosis.
sperm to move down the pistil to the female gametes Together, these observations led to the development of
(ova) below. the Chromosomal Theory of Inheritance, which
identified chromosomes as the genetic material
Plants used in first-generation crosses were called P0,
responsible for Mendelian inheritance.
or parental generation one, plants. Mendel collected
the seeds belonging to the P0 plants that resulted from The Chromosomal Theory of Inheritance was
each cross and grew them the following season. These consistent with Mendel’s laws and was supported by
offspring were called the F1, or the first filial (filial = the following observations:
offspring, daughter or son), generation. Once Mendel
examined the characteristics in the F1 generation of
plants, he allowed them to self-fertilize naturally. He
 • During meiosis, homologous chromosome pairs Homologous Recombination
migrate as discrete structures that are
In 1909, Frans Janssen observed chiasmata (the point
independent of other chromosome pairs.
at which chromatids are in contact with each other and
• The sorting of chromosomes from each
may exchange segments) prior to the first division of
homologous pair into pre-gametes appears to
meiosis. He suggested that alleles become unlinked
be random.
when chromosomes physically exchange segments.
• Each parent synthesizes gametes that contain
only half of their chromosomal complement. It is now known that the pairing and interaction
• Even though male and female gametes (sperm between homologous chromosomes, known as
and egg) differ in size and morphology, they synapsis, does more than simply organize the homologs
have the same number of chromosomes, for migration to separate daughter cells. When
suggesting equal genetic contributions from synapsed, homologous chromosomes undergo
each parent. reciprocal physical exchanges of DNA at their arms in a
• The gametic chromosomes combine during process called homologous recombination, or more
fertilization to produce offspring with the same simply, “crossing over.”
chromosome number as their parents.
Genetic Maps
It was only after several years of carrying out crosses
In 1913, Alfred Sturtevant, a student in Morgan’s
with the fruit fly, Drosophila melanogaster, that
laboratory, created the first “chromosome map,” a
Thomas Hunt Morgan provided experimental evidence
linear representation of gene order and relative distance
to support the Chromosomal Theory of Inheritance.
on a chromosome.To construct a chromosome map,
In 1910, Thomas Hunt Morgan started his work with Sturtevant assumed that genes were ordered serially on
Drosophila melanogaster, a fruit fly. He chose fruit flies threadlike chromosomes.
because they can be cultured easily, are present in
Sturtevant divided his genetic map into map units, or
large numbers, have a short generation time, and have
centimorgans (cM), in which a recombination frequency
only four pair of chromosomes that can be easily
of 0.01 corresponds to 1 cM.
identified under the microscope. They have three pair
of autosomes and a pair of sex chromosomes. At that Identification of Chromosomes and Karyotypes
time, he already knew that X and Y have to do with
A karyotype is the number and appearance of
gender. He used normal flies with red eyes and
chromosomes. To obtain a view of an individual’s
mutated flies with white eyes and cross bred them. In
karyotype, cytologists photograph the chromosomes
flies, the wild type eye color is red (XW) and is
and then cut and paste each chromosome into a chart,
dominant to white eye color (Xw). He was able to
or karyogram, also known as an ideogram.
conclude that the gene for eye color was on the X
chromosome. This trait was thus determined to be X- Chromosomes can be identified by their number, size,
linked and was the first X-linked trait to be identified. centromere position, and banding pattern. In a human
Males are said to be hemizygous, in that they have only karyotype, autosomes or “body chromosomes” (all of
one allele for any X-linked characteristic. the non–sex chromosomes) are generally organized in
approximate order of size from largest (chromosome 1)
to smallest (chromosome 22). However, chromosome
Genetic Linkage and Distances 21 is actually shorter than chromosome 22. This was
discovered after the naming of Down syndrome as
Mendel’s work suggested that traits are inherited
trisomy 21, reflecting how this disease results from
independently of each other. Morgan identified a 1:1
possessing one extra chromosome 21 (three total). The
ratio between a segregating trait and the X
X and Y chromosomes are not autosomes and are
chromosome, suggesting that the random segregation
referred to as the sex chromosomes.
of chromosomes was the physical basis of Mendel’s
model.
One such powerful cytological technique is karyotyping, with a dye to visualize the distinct banding patterns of
a method in which traits characterized by each chromosome pair.
chromosomal abnormalities can be identified from a
A karyotype can be used to visualize abnormalities in
single cell. To observe an individual’s karyotype, a
the chromosomes, such as an incorrect number of
person’s cells (like white blood cells) are first collected
chromosomes, deletions, insertions, or translocations
from a blood sample or other tissue. In the laboratory,
of DNA.
the isolated cells are stimulated to begin actively
dividing. A chemical called colchicine is then applied to
cells to arrest condensed chromosomes in metaphase.
Cells are then made to swell using a hypotonic solution Disorders in Chromosome Number
so the chromosomes spread apart. Finally, the sample is Aneuploidy, an abnormal number of chromosomes in a
preserved in a fixative and applied to a slide. cell, is caused by nondisjunction, or the failure of
A common stain choice is the Giemsa stain. Giemsa chromosomes to separate at meiosis.
staining results in approximately 400–800 bands (of Key Points
tightly coiled DNA and condensed proteins) arranged
along all of the 23 chromosome pairs. Aneuploidy is caused by nondisjunction, which occurs
when pairs of homologous chromosomes or sister
At its most basic, the karyotype may reveal genetic chromatids fail to separate during meiosis.
abnormalities in which an individual has too many or
too few chromosomes per cell. Examples of this are The loss of a single chromosome from a diploid genome
Down Syndrome, which is identified by a third copy of is called monosomy (2n-1), while the gain of one
chromosome 21, and Turner Syndrome, which is chromosome is called trisomy (2n+1).
characterized by the presence of only one X If homologous chromosomes fail to separate during
chromosome in women instead of the normal two. meiosis I, the result is no gametes with the normal
Geneticists can also identify large deletions or insertions number (one) of chromosomes.
of DNA. For instance, Jacobsen Syndrome, which
involves distinctive facial features as well as heart and If sister chromatids fail to separate during meiosis II, the
bleeding defects, is identified by a deletion on result is two normal gametes each with one copy of the
chromosome 11. Finally, the karyotype can pinpoint chromosome, and two abnormal gametes in which one
translocations, which occur when a segment of genetic carries two copies and the other carries none.
material breaks from one chromosome and reattaches
Aneuploidy can be lethal or result in serious
to another chromosome or to a different part of the
developmental disorders such as Turner Syndrome (X
same chromosome. Translocations are implicated in
monosomy) or Downs Syndrome (trisomy 21).
certain cancers, including chronic myelogenous
leukemia. Misaligned or incomplete synapsis, or a dysfunction of
the spindle apparatus that facilitates chromosome
Key Points
migration, can cause nondisjunction
A normal human karyotype contains 23 pairs of
Nondisjunction can occur during either meiosis I or II,
chromosomes: 22 pairs of autosomes and 1 pair of sex
with differing results. If homologous chromosomes fail
chromosomes, generally arranged in order from largest
to separate during meiosis I, the result is two gametes
to smallest.
that lack that particular chromosome and two gametes
The short arm of a chromosome is referred to as the p with two copies of the chromosome.
arm, while the long arm is designated the q arm.
Chromosomal Structural Rearrangements
To observe a karyotype, cells are collected from a blood
Structural rearrangements of chromosomes include
or tissue sample and stimulated to begin dividing; the
both inversions and translocations, which may have
chromosomes are arrested in metaphase, preserved in a
detrimental effects on an organism.
fixative and applied to a slide where they are stained
 Chromosome Inversions Early in development, when female mammalian
embryos consist of just a few thousand cells (relative to
A chromosome inversion is the detachment, 180°
trillions in the newborn), one X chromosome in each cell
rotation, and reinsertion of part of a chromosome.
inactivates by tightly condensing into a quiescent
Inversions may occur in nature as a result of
(dormant) structure called a Barr body. The chance that
mechanical shear, or from the action of transposable
an X chromosome (maternally or paternally derived ) is
elements (special DNA sequences capable of
inactivated in each cell is random, but once the
facilitating the rearrangement of chromosome
inactivation occurs, all cells derived from that single cell
segments with the help of enzymes that cut and paste
will have the same inactive X chromosome or Barr body.
DNA sequences).
By this process, a phenomenon called dosage
An inversion can be pericentric and include the
compensation is achieved. Females possess two X
centromere, or paracentric and occur outside of the
chromosomes, while males have only one; therefore, if
centromere. A pericentric inversion that is asymmetric
both X chromosomes remained active in the female,
about the centromere can change the relative lengths
they would produce twice as much product from the
of the chromosome arms, making these inversions
genes on the X chromosomes as males.
easily identifiable.
So how does X-inactivation help alleviate the effects of
The pericentric chromosome 18 inversion is believed to
extra X chromosomes? An individual carrying an
have occurred in early humans following their
abnormal number of X chromosomes will inactivate all
divergence from a common ancestor with chimpanzees
but one X chromosome in each of her cells.
approximately five million years ago. Researchers
characterizing this inversion have suggested that Individuals with three X chromosomes, called triplo-X,
approximately 19,000 nucleotide bases were are phenotypically female, but express developmental
duplicated on 18p, and the duplicated region inverted delays and reduced fertility. The XXY genotype,
and reinserted on chromosome 18 of an ancestral corresponding to one type of Klinefelter syndrome,
human. corresponds to phenotypically male individuals with
small testes, enlarged breasts, and reduced body hair.
Translocations
More complex types of Klinefelter syndrome exist in
A translocation occurs when a segment of a which the individual has as many as five X
chromosome dissociates and reattaches to a different, chromosomes.
nonhomologous chromosome. Translocations can be
Turner syndrome, characterized as an X0 genotype (i.e.,
benign or have devastating effects depending on how
only a single sex chromosome), corresponds to a
the positions of genes are altered with respect to
phenotypically female individual with short stature,
regulatory sequences.
webbed skin in the neck region, hearing and cardiac
Reciprocal translocations result from the exchange of impairments, and sterility.
chromosome segments between two nonhomologous
chromosomes such that there is no gain or loss of
genetic information. Duplications and Deletions

Duplications and deletions often produce offspring that

survive but exhibit physical and mental abnormalities.
X-Inactivation
Cri-du-chat (from the French for “cry of the cat”) is a
The presence of extra X chromosomes in a cell is
syndrome associated with nervous system
compensated for by X-inactivation in which all but one
abnormalities and identifiable physical features that
X chromosome are silenced.
result from a deletion of most of 5p (the small arm of
Rather than a gain or loss of autosomes, variations in chromosome 5). Infants with this genotype emit a
the number of X chromosomes are associated with characteristic high-pitched cry on which the disorder’s
relatively mild effects. In part, this occurs because of a name is based.
molecular process called X inactivation.
 Discovery of DNA Modern Applications of DNA

Deoxyribonucleic acid (DNA) is a molecule that carries The acronym “DNA” has become synonymous with
most of the genetic instructions used in the solving crimes, testing for paternity, identifying human
development, functioning and reproduction of all remains, and genetic testing. DNA can be retrieved
known living organisms and many viruses. from hair, blood, or saliva.

DNA is a nucleic acid; alongside proteins and Cloning

carbohydrates, nucleic acids are one of the three major
Reproductive cloning is a method used to make a clone
macromolecules essential for all known forms of life.
or an identical copy of an entire multicellular organism.
DNA stores biological information and is involved in
the expression of traits in all living organisms. In cloning both the original organism and the clone have
identical DNA.
The Path to Discovery
Cloning became an issue in scientific ethics when a
In the 1950s, Francis Crick and James Watson worked
sheep became the first mammal cloned from an adult
together to determine the structure of DNA at the
cell in 1996.
University of Cambridge, England. At the time, other
scientists like Linus Pauling and Maurice Wilkins were CRISPR
also actively exploring this field. Pauling had discovered
the secondary structure of proteins using X-ray CRISPR (Clustered, Regularly-Interspaced Short
crystallography. Palindromic Repeats) allows scientists to edit genomes,
far better than older techniques for gene splicing and
Chargaff’s Rule editing. The CRISPR technique has enormous potential
application, including altering the germline of humans,
Erwin Chargaff met Francis Crick and James D. Watson
animals and other organisms, and modifying the genes
at Cambridge in 1952, and, despite not getting along
of food crops.
with them personally, he explained his findings to
them. Chargaff’s Rule showed that in natural DNA, the Genetically Modified Organisms
number of guanine units equals the number of cytosine
units and the number of adenine units equals the A genetically modified organism (GMO) is any
number of thymine units. This strongly hinted towards organism whose genetic material has been altered
the base pair makeup of the DNA. Chargaff’s research using genetic engineering techniques. GMOs are a
would help the Watson and Crick laboratory team to source of medicines and genetically modified foods
deduce the double helical structure of DNA. and are also widely used in scientific research, along
with the production of other goods.
Franklin and X-Ray Diffraction
Genetic modification involves the mutation, insertion,
In Wilkins’ lab, researcher Rosalind Franklin used X-ray or deletion of genes.
diffraction methods to understand the structure of
DNA. Watson and Crick were able to piece together the
puzzle of the DNA molecule on the basis of Franklin’s The Structure and Sequence of DNA
data, because Crick had also studied X-ray diffraction. In
1962, James Watson, Francis Crick, and Maurice DNA is a double helix of two anti-parallel,
Wilkins were awarded the Nobel Prize in Medicine. complementary strands having a phosphate-sugar
backbone with nitrogenous bases stacked inside.
Unfortunately by then, Franklin had died. Nobel prizes
are not awarded posthumously, and though her work The monomeric building blocks of DNA are
was crucial to the discovery of DNA, Franklin was never deoxyribomononucleotides (usually referred to as just
nominated for a Nobel Prize. Francis Crick, James nucleotides), and DNA is formed from linear chains, or
Watson, and Maurice Wilkins were awarded a Nobel polymers, of these nucleotides.
Prize for the discovery of the structure of DNA in 1962.
The components of the nucleotide used in DNA developed. The most popular, widely-used second-
synthesis are a nitrogenous base, a deoxyribose, and a generation sequencing method was one called
phosphate group. Pyrosequencing.

The nitrogenous base can be a purine such as adenine In Illumina sequencing, up to 500,000,000 separate
(A) and guanine (G), characterized by double-ring sequencing reactions are run simultaneously on a
structures, or a pyrimidine such as cytosine (C) and single slide (the size of a microscope slide) put into a
thymine (T), characterized by single-ring structures. In single machine.
polynucleotides (the linear polymers of nucleotides)
Shotgun Sequencing
the nucleotides are connected to each other by covalent
bonds known as phosphodiester bonds or Most genomic sequencing projects today make use of
phosphodiester linkages. an approach called whole genome shotgun sequencing.
whole genome shotgun sequencing
The diameter of the DNA double helix is 2 nm and is
uniform throughout. Only the pairing between a purine Whole genome shotgun sequencing involves isolating
and pyrimidine can explain the uniform diameter. many copies of the chromosomal DNA of interest. The
chromosomes are all fragmented into sizes small
DNA sequencing is the process of determining the
enough to be sequenced (a few hundred basepairs) at
precise order of nucleotides within a DNA molecule.
random locations.
Knowledge of DNA sequences has become
indispensable for basic biological research, and in
numerous applied fields such as diagnostics, Basics of DNA Replication
biotechnology, forensic biology, and biological
systematics. DNA replication uses a semi-conservative method that
results in a double-stranded DNA with one parental
strand and a new daughter strand.
DNA Sequencing Techniques Models of Replication
The Sanger sequencing method was used for the There were three models of replication possible from
human genome sequencing project, which was finished such a scheme: conservative, semi-conservative, and
its sequencing phase in 2003, but today both it and the dispersive. In conservative replication, the two original
Gilbert method have been largely replaced by better DNA strands, known as the parental strands, would re-
methods. basepair with each other after being used as templates
to synthesize new strands; and the two newly-
Sanger Sequencing
synthesized strands, known as the daughter strands,
The Sanger method is also known as the dideoxy chain would also basepair with each other; one of the two
termination method. This sequencing method is based DNA molecules after replication would be “all-old” and
on the use of chain terminators, the the other would be “all-new”. In semi-conservative
dideoxynucleotides (ddNTPs). The dideoxynucleotides, replication, each of the two parental DNA strands
or ddNTPSs, differ from deoxynucleotides by the lack of would act as a template for new DNA strands to be
a free 3′ OH group on the five-carbon sugar. synthesized, but after replication, each parental DNA
strand would basepair with the complementary newly-
A Sanger sequencing reaction is just a modified in vitro
synthesized strand just synthesized, and both double-
DNA replication reaction.
stranded DNAs would include one parental or “old”
Second Generation and Next-generation Sequencing strand and one daughter or “new” strand. In dispersive
replication, after replication both copies of the new
The Sanger and Gilbert methods of sequencing DNA DNAs would somehow have alternating segments of
are often called “first-generation” sequencing because parental DNA and newly-synthesized DNA on each of
they were the first to be developed. In the late 1990s, their two strands.
new methods, called second-generation sequencing
methods, that were faster and cheaper, began to be
To determine which model of replication was accurate, a It also requires a free 3′-OH group to which it can add
seminal experiment was performed in 1958 by two nucleotides by forming a phosphodiester bond between
researchers: Matthew Meselson and Franklin Stahl. the 3′-OH end and the 5′ phosphate of the next
nucleotide. This means that it cannot add nucleotides if
Meselson and Stahl
a free 3′-OH group is not available. Another enzyme,
Meselson and Stahl were interested in understanding RNA primase, synthesizes an RNA primer that is about
how DNA replicates. They grew E. coli for several five to ten nucleotides long and complementary to the
generations in a medium containing a “heavy” isotope DNA, priming DNA synthesis. A primer provides the free
of nitrogen (15N) that is incorporated into nitrogenous 3′-OH end to start replication. DNA polymerase then
bases and, eventually, into the DNA. extends this RNA primer, adding nucleotides one by one
that are complementary to the template strand.
The DNA harvested from cells grown for two
generations in 14N formed two bands: one DNA band
was at the intermediate position between 15N and 14N
DNA Replication in Eukaryotes
and the other corresponded to the band of exclusively
14N DNA. These results could only be explained if DNA DNA replication in eukaryotes occurs in three stages:
replicates in a semi-conservative manner. initiation, elongation, and termination, which are aided
by several enzymes.

Initiation
DNA Replication in Prokaryotes
Eukaryotic DNA is bound to proteins known as histones
DNA replication employs a large number of proteins
to form structures called nucleosomes. During
and enzymes, each of which plays a critical role during
initiation, the DNA is made accessible to the proteins
the process. One of the key players is the enzyme DNA
and enzymes involved in the replication process. There
polymerase, which adds nucleotides one by one to the
are specific chromosomal locations called origins of
growing DNA chain that are complementary to the
replication where replication begins.
template strand.
Elongation
In prokaryotes, three main types of polymerases are
known: DNA pol I, DNA pol II, and DNA pol III. DNA pol During elongation, an enzyme called DNA polymerase
III is the enzyme required for DNA synthesis; DNA pol I adds DNA nucleotides to the 3′ end of the newly
and DNA pol II are primarily required for repair. synthesized polynucleotide strand. The template strand
specifies which of the four DNA nucleotides (A, T, C, or
There are specific nucleotide sequences called origins
G) is added at each position along the new chain. Only
of replication where replication begins. In E. coli, which
the nucleotide complementary to the template
has a single origin of replication on its one chromosome
nucleotide at that position is added to the new strand.
(as do most prokaryotes), it is approximately 245 base
pairs long and is rich in AT sequences. The origin of Termination
replication is recognized by certain proteins that bind to
Eukaryotic chromosomes have multiple origins of
this site. An enzyme called helicase unwinds the DNA
replication, which initiate replication almost
by breaking the hydrogen bonds between the
simultaneously. Each origin of replication forms a
nitrogenous base pairs. ATP hydrolysis is required for
bubble of duplicated DNA on either side of the origin of
this process. As the DNA opens up, Y-shaped structures
replication. Eventually, the leading strand of one
called replication forks are formed. Two replication
replication bubble reaches the lagging strand of another
forks at the origin of replication are extended bi-
bubble, and the lagging strand will reach the 5′ end of
directionally as replication proceeds. Single-strand
the previous Okazaki fragment in the same bubble.
binding proteins coat the strands of DNA near the
replication fork to prevent the single-stranded DNA
from winding back into a double helix. DNA polymerase
is able to add nucleotides only in the 5′ to 3′ direction (a
new DNA strand can be extended only in this direction).
 Telomere Replication Mutations, variations in the nucleotide sequence of a
genome, can also occur because of damage to DNA.
Key Points
Such mutations may be of two types: induced or
DNA polymerase cannot replicate and repair DNA spontaneous. Induced mutations are those that result
molecules at the ends of linear chromosomes. from an exposure to chemicals, UV rays, X-rays, or
some other environmental agent. Spontaneous
The ends of linear chromosomes, called telomeres, mutations occur without any exposure to any
protect genes from getting deleted as cells continue to environmental agent; they are a result of natural
divide. reactions taking place within the body.
The telomerase enzyme attaches to the end of the Some mutations are not expressed; these are known as
chromosome; complementary bases to the RNA silent mutations. Point mutations are those mutations
template are added on the 3′ end of the DNA strand. that affect a single base pair. The most common
Once the lagging strand is elongated by telomerase, nucleotide mutations are substitutions, in which one
DNA polymerase can add the complementary base is replaced by another. These can be of two types:
nucleotides to the ends of the chromosomes and the transitions or transversions. Transition substitution
telomeres can finally be replicated. refers to a purine or pyrimidine being replaced by a
base of the same kind; for example, a purine such as
Cells that undergo cell division continue to have their adenine may be replaced by the purine guanine.
telomeres shortened because most somatic cells do not Transversion substitution refers to a purine being
make telomerase; telomere shortening is associated replaced by a pyrimidine or vice versa; for example,
with aging. cytosine, a pyrimidine, is replaced by adenine, a purine.
Telomerase reactivation in telomerase-deficient mice Mutations can also be the result of the addition of a
causes extension of telomeres; this may have potential base, known as an insertion, or the removal of a base,
for treating age-related diseases in humans. known as a deletion. Sometimes a piece of DNA from
Key Terms one chromosome may get translocated to another
chromosome or to another region of the same
telomere: either of the repetitive nucleotide sequences chromosome.
at each end of a eukaryotic chromosome, which protect
the chromosome from degradation

telomerase: an enzyme in eukaryotic cells that adds a

specific sequence of DNA to the telomeres of
chromosomes after they divide, giving the
chromosomes stability over time

DNA Repair

Errors during Replication

DNA replication is a highly accurate process, but

mistakes can occasionally occur as when a DNA
polymerase inserts a wrong base. Uncorrected mistakes
may sometimes lead to serious consequences, such as
cancer.

Some errors are not corrected during replication, but

are instead corrected after replication is completed; this
type of repair is known as mismatch repair.