Subject FORENSIC SCIENCE

Paper No and Title DNA Forensics

Module No and Title DNA Structure and Function

Module Tag FS_P13_M1_ DNA structure and function

FORENSIC SCIENCE PAPER No. : 13 : DNA Forensics

MODULE No. : 1 DNA Structure and Function
 TABLE OF CONTENTS

1. Learning Outcomes
2. Introduction
3. Properties of DNA
3.1 Nitrogenous Bases
3.2 Grooves
3.3 Base pairing
4. Types of DNA
5. DNA Supercoiling
6. Summary

FORENSIC SCIENCE PAPER No. : 13 : DNA Forensics

MODULE No. : 1 DNA Structure and Function
 1. Learning Outcomes

After studying this module, you shall be able to

• Know the importance of DNA in living system.

• Learn the assembly of DNA and its constituents.
• Identify various types of physiological modifications in the assembly of DNA

2. Introduction

Deoxyribonucleic acid (DNA) is the primary genetic substance of living organisms and
some viruses. It is a major macromolecule that carries information important for every
forms of life. This information in DNA is transcripted to RNA and then further coded to
proteins for performing functions of an organism. Most DNA consists of two
biopolymers strands coiled around each other to form a double helix. DNA are
polynucleotides formed of two complementary strands that are twisted round each other
as double helical structure. The two strands are formed of monomers and are known as
nucleotides. Each nucleotide in turn contains one of four nitrogenous base; guanine (G),
adenine (A), thymine (T), or cytosine (C); 2’-deoxyribose which is a monosaccharide
sugar and a phosphate group. These nucleotides in the double helical structure are placed
near by covalent bonds between -OH group of sugar of one nucleotide and the phosphate
of the subsequently, resultant in a sugar-phosphate backbone. Two strands in DNA are
placed near each other as per Chargaff’s rule, (A always pairs with T and C pairs with G
with hydrogen bonds).

DNA

Nucleus

Prokaryotic Cell Eukarotic Cell

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 Figure 1. DNA in a cell. DNA is structured into a single
circular molecule in prokaryotic cells like bacteria where as in eukaryotes it is present in form of
multiple thread like chromosomes inside a membrane bound nucleus.

Within living cells, DNA is structured into extended thread like arrangement called
chromatin that condenses to form chromosomes during Mitosis or Meiosis. Prior to cell
division, the DNA content of the cell is duplicated by progression of DNA replication, in
case both daughter cells with its individual whole set of chromosomes. In Eukaryotic
organisms (those which have nucleated cells such as animals and plants) DNA is present
inside the nucleus and a small proportion in semiautonomous organelles like as
mitochondria or chloroplasts. In contrast, prokaryotes (primitive cells without nucleus
such as bacteria and archaea) store their DNA in the protoplasm (Figure 1). Contained by
the cell, DNA is allied with proteins such as histones or polyamines that make DNA
compact and help in higher orders of its organization. These higher order structures
(called nucleosomes) stabilize the DNA and helps in modulating control of synthesis and
interactions between DNA, RNA and Proteins.

The polymeric nature of DNA was understood in the late 1930s but it is in the year 1953
that D S Watson and F H Cricked presented working model of DNA by obtaining data
tea, two source- base composition of hydrolysed, samples of DNA and X-Ray diffraction
studies of DNA.

During 1950s, Erwin Chargaff and his colleagues answered the composition of DNA
from various organisms by using chromatographic methods. The following conclusions
were drawn from the data:
1. The amount of purine residue is proportional to the amount of pyrimidine residues of
DNA of any species.
2. The amount of adenine (A) is proportional to the amount of thymine (T) and of
cytosine is equal to guanine (G).
3. The percentage of A+T does not necessarily equal to the percentage of G+C. The ratio
although varies greatly among species is constant for particular species.
The quantitative relationship given by Chargaff is known as Chargaff’s rule and is
universally applicable to all species. X-rays diffraction analysis of DNA was first carried
out by William Astbury in 1938 and highlighted the presence of repeating units of 3.4 A⁰
in DNA in 1947. Later it was Rocabid Franklin and Munrice Wilkins in 1950 who
showed that DNA produce a characteristic X- ray diffraction pattern and concluded that
DNA is helix with two periodicities along the long axis one at o.34 nm and other at 3.4
nm respectively.

FORENSIC SCIENCE PAPER No. : 13 : DNA Forensics

MODULE No. : 1 DNA Structure and Function
 Watson and Cricked in 1953 proposed a three-dimensional mode of DNA in the
following main features:
1. Two helical DNA chains coiled around a long axis forming a right/left handed
double helix.
2. The two chains are anti-parallel. That is there C-5’ to C-3’ orientations run in
opposite directions. The two chains cannot be separated without unwinding the helix, a
phenomenon known as plectonemic coiling.
3. The bases of the chains are flat structure arranged perpendicular to the long axis.
They are stacked on one another at a distance of 0.34 nm.
4. The anti-parallel chains of DNA are complementary and not identical. The
purines and pyrimidines in both the chains are paired to one another by the formation of
hydrogen bonds. A pair with T by two hydrogen bands and G with C by three hydrogen
bonds.

Figure 2. Double helical structure of DNA as given by Watson and Crick (1953).
(a) 2 strands of DNA are complementary to each other and A pair with T and G pairs
with C by double and triple hydrogen bonds respectively. (b) The two strands are anti-
parallel [Link] in opposite direction.

FORENSIC SCIENCE PAPER No. : 13 : DNA Forensics

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 DNA is a highly stable molecule and it is therefore suitable for the storage of biological
information. The sugar-phosphate backbone of DNA is impervious to hydrolysis.
Moreover, the 2 strands of the double helix impart the same genetic information due to
their complementarity. Bulk of DNA within eukaryotic cells is non-coding and do not
serve a function of encoding proteins. This may amount to approximately 98% in human
cells.

The 2 strands of DNA are antiparallel; running in reverse directions with respect to each
other - one backbone being 3′ and the further 5′. Joined to each sugar is either a purine or
pyrimidine nitrogenous base aligned in a sequence that beside the backbone encrypts
biological information. Information from DNA is transcripted to RNA which further
translates it to protein molecules that are the true effector molecules of genetic info.

3. Discovery of 3d Structure

DNA is a hetero-polymer of deoxyribonucleotides. DNA was primary identified and

taken out by Friedrich Miescher. However the structure and functions of DNA remained
obscured for several decades. James D Watson and Francis Crick gave the model for
double helix assembly of DNA in 1953. The arrangement of DNA of all classes is
remarkably identical and comprises two antiparallel helical chains each twisted round the
similar axis, and individually with a pitch of 34 ångströms (3.4 nanometres) and a radius
of 10 ångströms (1.0 nanometres) and one nucleotide unit measured 3.3 Å (0.33 nm)
long. Although every discrete repeating unit is very minor, DNA polymers can be very
big molecules comprising millions of nucleotides. For example, the biggest human
chromosome, chromosome number 1, comprises of around 220 million base pairs and is
about 85 nm in length.

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 5’ 3’ 5’ 3’

Figure 3. Structure of DNA. 2 strands of DNA are entwined in a helical manner and run
antiparallel to each other. Purines A and G are paired with Pyrimidines C and T through hydrogen
bonding against a sugar phosphate backbone. Note that helical structure of DNA gives rise to
formation of major and minor grooves. Image Source: Department of Biology- Penn State
University.

Within living systems, DNA occurs as a pair of molecules that are placed firmly close by
hydrogen bonding between the nitrogenous bases; the strands are entwined as a double
helix. The nucleotide repeats comprise together the section of the backbone of the
molecule, that place the chain close, and a nitrogenous base, it networks with the other
DNA strand in the helix. A nitrogenous base linked to a sugar is known as nucleoside and
a base linked to a sugar and one or more phosphate groups is known as nucleotide. A
polymer containing multiple connected nucleotides (as in DNA) is known as
polynucleotide.

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 The support of each individual DNA strand contains alternate phosphate and 2’-
deoxyribose sugar residues that are joined by phosphodiester bonds among the 3rd and 5th
carbon atoms of nearby sugar rings. This specific bonding pattern makes each strand of
DNA double helix molecule directional and asymmetric. As a result, the two asymmetric
strands move in reverse direction and are antiparallel. The asymmetric terminals of DNA
strands are known as the 5′ and 3′ ends, with the 5′ end has a terminal phosphate group
and the 3′ end a terminal hydroxyl group. The double helix structure is primarily
stabilized by 2 forces: hydrogen bonds among nucleotides and base-stacking connections
between aromatic nitrogenous bases. In the aqueous environment of the cell, the
conjugated π bonds of nucleotide bases arrange vertical to the axis of the DNA molecule.
This alignment minimizes their interactions with the solvation shell and consequently, the
Gibbs free energy imparting stability to DNA.

3.1. Nitrogenous Bases

The nitrogenous bases are also called nucleobases are categorized into 2 forms: the
purines and pyrimidines. Purines- Adenine and Guanine; are merged five- and six-
membered heterocyclic compounds, and the pyrimidines- Cytosine and Thymine; are the
six-membered rings.

Figure 4: Nitrogenous Bases. Nitrogenous bases within DNA are classified into purines
and pyrimidines. Adenine and Guanine are Purines whereas Thymine and Cytosine are
Pyrimidine. In addition, Uracil is another pyrimidine formed from cytosine and is usually
present in RNA only.

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 A 5th pyrimidine uracil (U); is present in position of thymine in RNA and is not found in
DNA.

3.2. Grooves

Identical helical strands that make the DNA backbone have been observed outlining the
spaces, or grooves, among the strands. These spaces are adjacent to the base pairs and
offer a binding site for proteins. Since 2 strands are asymmetrically situated in reference
with each other, the furrows are unequal in size. Larger of the 2 grooves; the major
groove, is 22 Å in span while the smaller; minor groove is 12 Å in width. This means that
the edges of the bases are more accessible in the major groove. As a result, proteins such
as transcription factors that can join to particular sequences in double-stranded DNA
generally forms connections to the edges of the bases open in the major groove.

Figure 5: DNA grooves. Due to the helical alignment of 2 antiparallel strands of DNA two
asymmetric grooves are formed. Wider of the 2 is called major groove and binds to a number of
key enzymes and proteins that regulate activity of DNA.

3.3. Base Pairing

In a DNA double helix, every form of nitrogenous base on one strand bonds with only
one form of nitrogenous base on the other strand. This property is known as
complementary base pairing. The purines form hydrogen bonds to pyrimidines, with
adenine bonding only to thymine by two hydrogen bonds, and cytosine bonding only to
guanine via three hydrogen bonds. This set of purine-pyrimidine nucleotides binding
together through the double helix is known as base pair. Since G-C base pair has one
additional hydrogen bond, it is more even in comparison to A-T base pair. Consequently,
DNA molecules with higher GC-content are relatively more stable.

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 Austrian chemist Erwin Chargaff discovered a pattern that DNA makes any cell of all
entities should have a 1:1 ratio (base Pair Rule) of purine and pyrimidine bases and, the
quantity of guanine and adenine is equivalent to that of cytosine and thymine. This is now
commonly known as Chargaff’s rule.

Figure 6: DNA Base Pairs. Purines and Pyrimidines in DNA are placed together by hydrogen
bonds. Adenine is bonded to Thymine through 2 hydrogen bonds whereas Guanine is linked with
cytosine via 3 hydrogen bonds.

The constancy of the double stranded DNA depends on the GC-content (% G-C
basepairs), length (longer molecules are more stable) as well as on sequence (stacking is
sequence specific). The stability of DNA can be known in terms of "melting
temperature", that is the temperature at which half (50%) of the double stranded
molecules are transformed to single stranded molecules; melting temperature is
dependent on ionic strength and the concentration of DNA. Under laboratory conditions,
the strength of contact can be calculated from the temperature essential to breakdown the
hydrogen bonds, their melting temperature (also called Tm value). Long DNA helices
with a more GC-content has harder-interacting strands, while small helices with more AT
content have weaker-interacting strands. Regions of the DNA double helix that require to
isolated easily, like as the TATAAT or Pribnow box in several promoters, incline to have
a high AT content, making the strands easier to separate and thus carry out biological
functions such as replication, transcription, DNA repair and regulation.

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 Since hydrogen bonds are not covalent and are weak, they can be fragmented and joined
again comparatively easily. The 2 strands of DNA in a double helix can consequently be
drawn apart such as zipper, either by a mechanical force or high temperature.

This property is valuable while assessing functional role of DNA and as a consequence of
this complementarity, all the info in the double-stranded arrangement of a DNA helix is
reproduced on every strand, which is important in DNA replication. This reversible and
precise interaction between complementary base pairs is important for all the functions of
DNA in living beings.

4. Types of DNA

Three chief kinds of double stranded DNA are known to exist. These are termed A, B and
Z-form DNA. The most usual form of DNA present at neutral pH and physiological salt
concentrations is B-form that is right-handed double helical assembly. A relatively denser
right-handed duplex with lesser distance among the base pairs has been labelled for
RNA-DNA duplexes and RNA-RNA duplexes. This is known A-form nucleic acid. In
contrast to right handed A and B- DNA, Z-form of duplex DNA has a extremely diverse,
left-handed helical assembly shaped by stretches of interchanging purines and
pyrimidines, e.g. GCGCGC, particularly in negatively supercoiled DNA. Only a small
proportion of cellular DNA exists in the Z form. It will be interesting to correlate that this
different structure is involved in some way in regulation of some cellular function, like as
transcription or regulation, but convincing evidence for or in contradiction this proposal
is not accessible yet. Table 1 shows the differences between three types of DNA.

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 A- DNA B- DNA Z- DNA
Figure 7: Structure of different forms of DNA. B DNA is most commonly present under
physiological conditions.

A-form B-form Z-form

It is the hydrated form of This is the most common It occurs in a small
the DNA. It occurs when form of DNA in living cells. proportion in cellular drift.
the relative humidity of B- It is a right handed helix. Its sugar-phosphate
backbone form a zig-zag
DNA is reduced to 75%.
pattern instead of a smooth
Under such conditions, B- helix, hence the name Z-
DNA undergoes a DNA. Z-DNA has 12
reversible conformational Watson and Crick base pair
change to form a flatter and per turn and a pitch of 45
wider right handed helix. It A⁰ and compare to A-DNA,
has a deep major groove a deep minor groove and no
distinct major groove.
and a shallow minor
groove.

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 Table -1: Comparison of different forms of DNA.
Helix Type
A B Z
Direction of helix Right handed Right handed Left handed
Shape Broad Intermediate Narrow
Helix Diameter 2.3 A 3.4 A 3.8 A
Rise per base pair 25.5 A 23.7A 18.4 A
Glycosidic Bond anti anti Alternate anti and syn
No. of bases/ turn 11 10.4 12
Pitch 25.3 A 35.4 A 45.6 A
Tilt from axis 19° 1° 9°
Major groove Narrow (deep) Wide (deep) Flat
Minor groove Wide (shallow) Narrow (deep) Narrow (deep)
Occurrence dehydration condition hydrated condition dehydrated high salt
concentration

syn and anti implies to alignment of the N-gycosidic linkage between deoxyribose and
nitrogenous base. In anti-orientation base extend away from the deoxyribose where as in
syn orientation, it is above the deoxyribose. Pyrimidine can only be in anti-orientation
whereas purines can be in either of the two.

5. DNA Supercoiling

Supercoiling of DNA was discovered by Vinograd and Coworkers in 1963. The coiling
of DNA could be of 2 types:
1. Positive coiling- when DNA molecule is coiled in a direction similar to the
direction of twisting of the two polynucleotides strands tens to overwind. This type is
called positive super coiling and is formed in eukaryotic chromosomes.
2. Negative supercoiling: when DNA molecule is coiled in a direction opposite to
the direction of twisting of duplex, the two strands tend to unwind. This type of coiling is
known as negative supercoiling. It is found in circular DNA present in bacterial viruses
and mitochondria.

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MODULE No. : 1 DNA Structure and Function
 6. Summary

• Deoxyribonucleic acid (DNA) is a biopolymer that contains and encrypts the

genetic information applied in the growth and functioning living organisms.
• DNA together with proteins and carbohydrates is one of the three chief
macromolecules.
• DNA is made up of 2 antiparallel strands that are coiled round each other to form
a double helix. The strands of DNA comprise of simpler units known as
nucleotides.
• Nucleotides are comprised of a nitrogenous base; purines - guanine (G) or adenine
(A) or prymidines- thymine (T), or cytosine (C) — as well as a monosaccharide
sugar called 2’-deoxyribose and a phosphate group.
• Nucleotides in DNA are linked to one another by phosphodiester covalent bonds
among the sugar of one nucleotide and the phosphate of the subsequent, resultant
in a sugar-phosphate backbone.
• The purines form hydrogen bonds to pyrimidines, with adenine forms bonding
with thymine by 2 hydrogen bonds, and cytosine bonding only to guanine via 3
hydrogen bonds.
• A set of purine-pyrimidine nucleotides bonded together around the double helix is
called as base pair.
• DNA is highly stable molecule making it suitable to store the biological
information.

FORENSIC SCIENCE PAPER No. : 13 : DNA Forensics

MODULE No. : 1 DNA Structure and Function