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What is DNA?
DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms.
Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it
is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is
called mitochondrial DNA or mtDNA).
The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G),
cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99 percent of
those bases are the same in all people. The order, or sequence, of these bases determines the information
available for building and maintaining an organism, similar to the way in which letters of the alphabet
appear in a certain order to form words and sentences.
DNA bases pair up with each other (Figure 21), A with T and C with G, to form units called base pairs.
Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and
phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a
double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the
ladder’s rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder.
• An important property of DNA is that it can
replicate, or make copies of itself. Each strand of
DNA in the double helix can serve as a pattern for
duplicating the sequence of bases. This is critical
when cells divide because each new cell needs to
have an exact copy of the DNA present in the old
cell.
DNA Double Heliex
Watson and Crick
Watson
Crick
The Roles of Nucleic Acids in heredity :
 There are two types of nucleic acids:


Deoxyribonucleic acid (DNA) and Ribonucleic
acid (RNA).
DNA provides direction for its own replication.
DNA also directs RNA synthesis and, through
RNA, controls protein synthesis.
Organisms inherit DNA from their parents:
 When a cell divides, its DNA is copied and
passed to the next generation of cells.
DNA Structure
DNA is a nucleic acid.
The building blocks of DNA are
nucleotides, each composed of:
a 5-carbon sugar called deoxyribose
 a phosphate group (PO4)
 a nitrogenous base

 adenine, thymine, cytosine, guanine
 Watson and Crick
G
C
A
deduced that DNA
was a double helix
T
T
1 nm
Through
observations of
the X-ray
crystallographic
images of DNA
C
G
C

A
3.4 nm
G
A
T
G
C
T
A
T
A
A
T
T
A
G
A
C
0.34 nm
T
(a) Key features of DNA structure
(c) Space-filling model
 Watson and Crick had concluded that DNA

Was composed of two antiparallel sugarphosphate backbones, with the nitrogenous
bases paired in the molecule’s interior
 The nitrogenous bases
Are paired in specific combinations:
 adenine A with thymine T, and cytosine C
with guanine G

5 end
O
OH
P
–O
Hydrogen bond
3 end
OH
O
O
O
O
O
P
–O
CH2
O
O
H2C
O
G
O–
P
O
O
O
C
O
O
P
–O
CH2
O
O
H2C
O
–O
A
T
O
O
C
O
G
O
O
P
O–
P
CH2
O
O
H2C
O
O
A
CH2
O
O
(b) Partial chemical structure
O
T
O
OH
3 end
O–
P
O–
P
O
5 end
 Watson and Crick reasoned that there
must be additional specificity of pairing
 Each base pair forms a different
number of hydrogen bonds

Adenine (A) and thymine (T) form two
bonds, cytosine (C) and guanine (G)
form three bonds
Nucleotides are connected to each other
to form a long chain
phosphodiester bond: bond between
adjacent nucleotides

formed between the phosphate group of
one nucleotide and the 3’ –OH of the
next nucleotide
The chain of nucleotides has a 5’ to 3’
orientation.
H
N
N
N
N
Sugar
CH3
O
H
H
N
N
N
O
Adenine (A)
Sugar
Thymine (T)
H
O
N
N
Sugar
N
H
H
N
N
N
N
N
Guanine (G)
H
H
O
Cytosine (C)
Sugar
 Many proteins work together in DNA

replication and repair
The relationship between structure and
function:

Is manifest in the double helix
 Since the two strands of DNA are
complementary

Each strand acts as a template for
building a new strand in replication
• The two strands are complementary
• e.g. If a segment of one strand has the base
•
sequence:
AGGTCCG
Then the same segment of the other strand
must
have the sequence:
TCCAGGC
DNA Structure
Determining the 3-dimmensional structure of DNA
involved the work of a few scientists:

Erwin Chargaff determined that
 amount of adenine = amount of thymine
 amount of cytosine = amount of guanine
This is known as Chargaff’s Rules
Sugar-phosphate
Backbone
Prior to the 1950s, it was already
known that DNA Is a polymer
of nucleotides, each consisting
of three components:
1- a nitrogenous base,
2- a sugar, and
3- a phosphate group
5 end
5
CH2
O P O
Nitrogenous
bases
CH3
O–
O–
4
H
O
1
H
H
3
2
H
CH2
P O
O–
H
O
Thymine (T)
H
H
H
H
H
Adenine (A)
H
H
CH2
P O
O–
H
H
O
H
H
N H
N
H
5
CH2
O
1
4 H
H
Phosphate H
H
2
3
H
OH
Sugar (deoxyribose)
3 end
P O
O–
N
O
Cytosine (C)
H
O
H
N
N
O
O
N
N
H
O
H
N
O
H
N
N
H
O
O
O
H
H
N
O
N
N
N H
N H
H
Guanine (G)
DNA nucleotide
DNA Double
Helix
AP Biology
DNA Structure
The double helix consists of:
2 sugar-phosphate backbones
 nitrogenous bases toward the interior of
the molecule
 bases form hydrogen bonds with
complementary bases on the opposite
sugar-phosphate backbone
 G binds C by three hydrogen bonds
 A binds T by two hydrogen bonds

 a: General Lab safety in molecular biology
 In molecular biology lab a number of chemicals are used that are




hazardous and can cause severe burn and long term sickness requiring
immediate medical attention. Hence, before conducting an experiment it
is essential to you know the safety precautions and risk associated with
handling the chemical compound. The following chemicals are
especially noteworthy:
Phenol: Can cause severe burns
Acryl amide: potentially neurotoxins
Ethidium bromide: a strong carcinogen
In order to make sure the safe handling of the chemicals always follow
the following safety precautions:
 1.
 2.
 3.




Wear gloves while handling hazardous chemicals
Never mouth pipettes any chemicals
Always use fresh tips or pipette for each solution samples
to avoid contamination of the samples and the solutions.
4. If any chemical is accidently spilt on the skin, immediately
rinse with a lot of water and inform the instructor.
5. Always discard the waste in appropriate waste disposal as
instructed by the instructor.
Ultra violet light: UV lamp will be used to visualize the DNA
bands on the gel following electrophoresis. Direct exposure to
UV light can cause acute eye irritation and skin allergy. Since
retina cannot detect UV light, serious eye damage may be
caused if exposed to UV, therefore always wear safety goggles
or eye protection when using UV lamps.
Electricity: The voltage used for electrophoresis is sufficient to
cause electrocution. Cover the buffer reservoir during
electrophoresis and always switch off the power supply and
unplug the lead before removing the gel from electrophoresis
unit.
 General tips for conducting a safe and successful






experiment.
1. Always keep the work area clean of any unwanted
tubes, beakers and dirty dishes.
2. All reagents should be marked clearly with reagent
name and concentration.
3. All samples should be numbered and labeled
correctly with the names and dates.
4. Make sure that after use the reagents and chemical are
placed in the fridge or freezer as required.
5. In bacterial cultures make sure the reagents and dishes
are autoclaved properly and label using autoclave taps.
6. Always mark the bottom of the bacterial culture dishes
and not the lid, as the lids can easily be mixed up.

 3.1 Introduction
 There are different protocols and several commercially available
kits that can be used for the extraction of DNA from whole blood.
This procedure is one routinely used both in research and
clinical service provision and is cheap and robust. It can also be
applied to cell pellets from dispersed tissues or cell cultures
(omitting the red blood lysis step.
 3.2 Theory
 Successful nucleic acid isolation protocols have been published
for nearly all biological materials. They involve the physical and
chemical processes of tissue homogenisation (to increase the
number of cells or the surface area available for lysis), cell
permeabilisation, cell lysis (using hypotonic buffers), protein
degradation and removal of nucleases, protein precipitation,
solubilisation of nucleic acids and finally various washing steps.
Cell permeabilisation may be achieved with the help of non-ionic
(non DNA-binding) detergents such as SDS and Triton.
Mutation
A mutation is a permanent change in the DNA sequence of a
gene. Mutations in a gene's DNA sequence can alter the amino
acid sequence of the protein encoded by the gene.
How does this happen? Like words in a sentence, the DNA
sequence of each gene determines the amino acid sequence for
the protein it encodes. The DNA sequence is interpreted in
groups of three nucleotide bases, called codons. Each codon
specifies a single amino acid in a protein.
What is a gene mutation and how do mutations occur?
A gene mutation is a permanent change in the DNA sequence that makes
up a gene. Mutations range in size from a single DNA building block
(DNA base) to a large segment of a chromosome.
Gene mutations occur in two ways: they can be inherited from a parent
or acquired during a person’s lifetime. Mutations that are passed from
parent to child are called hereditary mutations or germline mutations
(because they are present in the egg and sperm cells, which are also
called germ cells). This type of mutation is present throughout a person’s
life in virtually every cell in the body.
Mutations that occur only in an egg or sperm cell, or those that occur
just after fertilization, are called new (de novo) mutations. De novo
mutations may explain
• genetic disorders in which an affected child has a mutation in every
cell, but has no family history of the disorder.
• Acquired (or somatic) mutations occur in the DNA of individual
cells at some time during a person’s life. These changes can be
caused by environmental factors such as ultraviolet radiation from
the sun, or can occur if a mistake is made as DNA copies itself
during cell division. Acquired mutations in somatic cells (cells other
than sperm and egg cells) cannot be passed on to the next generation.
• Mutations may also occur in a single cell within an early embryo. As
all the cells divide during growth and development, the individual
will have some cells with the mutation and some cells without the
genetic change. This situation is called mosaicism.
• Some genetic changes are very rare; others are common in the
population. Genetic changes that occur in more than 1 percent of the
population are called polymorphisms. They are common enough to be
considered a normal variation in the DNA. Polymorphisms are
responsible for many of the normal differences between people such
as eye color, hair color, and blood type. Although many
polymorphisms have no negative effects on a person’s health, some
of these variations may influence the risk of developing certain
disorders.
• The DNA sequence of a gene can be altered in a number of ways.
Gene mutations have varying effects on health, depending on where
they occur and whether they alter the function of essential proteins.
Misssense Mutation
This type of mutation is a change in one DNA base pair that results in the substitution of one
amino acid for another in the protein made by a gene (Figure 22).
Figure 22. Missense mutation. In this example, the nucleotide adenine is replaced by
cytosine in the genetic code, introducing an incorrect amino acid into the protein
sequence.
1. Nonsense mutation
A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino
acid for another, however, the altered DNA sequence prematurely signals the cell to stop building
a protein. This type of mutation results in a shortened protein that may function improperly or not
at all (Figure 23).
Figure 23. Monsense mutation. In this example, the nucleotide cytosine is replaced
by thymine in the DNA code, signaling the cell to shorten the protein.
3.Insertion
An insertion changes the number of DNA bases in a gene by adding a piece of
DNA. As a result, the protein made by the gene may not function properly
(Figure 24).
Figure 24. Insertion mutation. In this example, one nucleotide (adenine) is added in the DNA code,
changing the amino acid sequence that follows.
4.Deletion
A deletion changes the number of DNA bases by removing a piece of DNA. Small deletions may remove one or a few
base pairs within a gene, while larger deletions can remove an entire gene or several neighboring genes (Figure 25).
The deleted DNA may alter the function of the resulting protein(s).
Figure 25. Deletion mutation. In this example, one nucleotide (adenine) is deleted from the
DNA code, changing the amino acid sequence that follows.
•
5.Duplication
•
A duplication consists of a piece of DNA that is abnormally copied one or more times. This type of
mutation may alter the function of the resulting protein (Figure 26).
Figure 26. Duplication mutation. A section of DNA is accidentally duplicated when a
chromosome is copied.
6.Frameshift mutation
This type of mutation occurs when the addition or loss of DNA bases changes a gene’s reading
frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A
frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The
resulting protein is usually nonfunctional. Insertions, deletions, and duplications can all be
frameshift mutations (Figure 27).
Figure 27. Frameshift mutation. A frameshift mutation changes the amino acid
sequence from the site of the mutation.