GRADE: XII BIOLOGY

CH-6-MOLECULAR BASIS OF INHERITANCE[Study material]

Nucleic acids
 Nucleic acids are the macromolecules found in all living organisms.
 The two types of nucleic acids in living organisms are:
 Deoxyribonucleic acid (DNA)
 Ribonucleic acid(RNA)
The DNA
 DNA is a long polymer of deoxyribonucleotides. The length of DNA is
usually defined as the number of nucleotides present in it.
 Example: Bacteriophage lambda has 48502 base pairs (bp), Escherichia coli
has 4.6 × 106 bp, and haploid content of human DNA is 3.3 × 109 bp
Chemical Structure of Polynucleotide Chain (DNA/RNA) :
A nucleotide has three components.
(a) Nitrogen base
(i) Purines: Adenine and Guanine
(ii) Pyrimidines: Cytosine, Thymine and Uracil [Thymine in DNA and
Uracil in RNA].
(b) Pentose Sugar: Ribose (in RNA) or Deoxyribose (in DNA).
(c) Phosphate Group
 Nitrogen base is linked to pentose sugar through N-Glycosidic linkage.
Nitrogen base + Sugar = Nucleoside
 Phosphate group is linked to 5’-OH of a nucleoside through phosphoester
linkage.
Nucleoside + Phosphate group = Nucleotide
 Two nucleotides are linked through 3’-5 phosphodiester linkage to form a
Dinucleotide
 A polynucleotide chain has free phosphate group at 5’ end of ribose sugar

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 and a free 3’-OH group at the other end.
 RNA is highly reactive than DNA: In RNA nucleotide has an additional
OH group at 2’ positions in the ribose; RNA is also catalytic.
Double-helix Structure of DNA: Proposed by Watson and Crick in 1953
(i) DNA is made up of two polynucleotide chains.
(ii) The backbone is made up of sugar and phosphate and the bases project
inside.
(iii) Both polynucleotide chains are antiparallel i.e. one chain has polarity
5’-3’ and the other chain has 3’-5’.
(iv)These two strands of chains are held together by hydrogen bonds i.e.A = T,
C G.
(v) Both chains are coiled in right-handed fashion. The pitch of helix is 3.4 nm
with 10 base pairs in each turn. Consequently, the distance between a bp in a
helix is approximately equal to 0.34 nm.
(vi)The plane of one base pair stack over the other in double helix. This, in
addition to H-bonds, confers stability of the helical structure.

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Central dogma in molecular biology
 Francis Crick proposed the Central dogma in molecular biology, which
states that the genetic information flows from DNARNAProtein.

 In some viruses(eg: HIV) the flow of information is in reverse direction, that

is, from RNA to DNA. This is known as reverse transcription.
Packaging of DNA Helix
 The average distance between the two adjacent base pairs is 0.34
nm (0.34 × 10-9or 3.4°A)
 The number of base pairs in Escherichia coli is 4.6 ×106.
DNA Packaging in Prokaryotes:
DNA is not scattered throughout the cell. DNA (negatively Charged) is held by
some proteins (has positive charges) in a region termed as nucleoid. The DNA
in nucleoid is organized in large loops held by proteins.
DNA packaging in Eukaryotes:
 There is a set of positively charged basic proteins called histones. Eight
histone molecules combines together to form histone octamer.
 The negatively charged DNA is wrapped around positively charged
histone octamer to form as structure called nucleosome.
 Histone H1 is situated outside of nucleosomal DNA in linker region.
 Nucleosomes constitute the repeating unit of a structure in nucleus called
Chromatin
 The beads-on-string structure in chromatin is packaged to form chromatin
fibres that are further coiled and condensed at metaphase stage of cell
division to form chromosomes.
 The packaging of chromatin at higher level requires additional set of
protein that collectively are referred to as non-histone chromosomal
(NHC) proteins. At places chromatin is density packed to form darkly
staining heterochromatin. At other places chromatin is loosely packed to
form euchromatin.
 Euchromatin is said to be transcriptionally active chromatin, whereas.
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 Heterochromatin is inactive.
Transforming Principle:
Frederick Griffith (1928) performed experiments with Streptococcus
phenumoniae and mice. This bacterium has two strains.
1. S-strain (Virulent)-which possess a mucilage coat and has ability to cause
pneumonia.
2. R-strain (Nonvirulent) which do not possess mucilage coat and is unable
to cause pneumonia.
 Griffth injected R-strain bacteria into mice.
 No disease noticed and mice remain live.
 On injecting S-strain bacteria into mice.
Mice died due to pneumonia.
 When heat-killed S-strain bacteria were injected into mice  No
pneumonia symptoms noticed and mice remain live.
 He then injected a mixture of R-strain bacteria (Non virulent) and heat
killed S-strain bacteria (virulent) into mice.  mice died due to
pnuemonia.
 Moreover, Griffith recovered living S-strain (virulent) bacteria from the
dead mice.
Conclusion: He concluded that presence of heat-killed S-strain bacteria caused
transformation of some R-strain bacteria into virulent by a chemical substance,
called “transforming principle”.But biochemical nature of the genetic material was
not defined by him.
Chemical Nature of Transforming Principle
 In 1944, Avery MacLeod and McCarty worked to determine the chemical
nature of ‘transforming principle’.
 They purified biochemicals (proteins, DNA, RNA, etc.) from the heat-killed
S cells to see which ones could transform live R cells into S cells.
 They also discovered that protein-digesting enzymes (proteases) and RNA-
digesting enzymes (RNases) did not affect transformation, so the
transforming substance was not a protein or RNA.
 Digestion with DNase did inhibit transformation, suggesting that the DNA
caused the transformation.
Hershey and Chase Experiment: In 1952, Hershey and Chase performed an
experiment on bacteriophages (Viruses that infect bacteria) and proved that
DNA is the genetic material.
 They grew some viruses on a medium that contained radioactive phosphorus
and some others on medium that contained radioactive sulfur.

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 Viruses grown in the presence of radioactive phosphorus contained
radioactive DNA but not radioactive protein because DNA contains
phosphorus, but protein does not.
 Similarly, viruses grown on radioactive sulfur contained radioactive protein
but not radioactive DNA because DNA does not contain sulfur.
 Radioactive phages were allowed to attach to E. coli bacteria.
 Then, as the infection proceeded, the viral coats were removed from the
bacteria by agitating them in a blender.
 The virus particles were separated from the bacteria by spinning them in a
centrifuge.
 Bacteria which were infected with viruses that had radioactive DNA were
radioactive, indicating that DNA was the material that passed from the virus
to the bacteria
 Bacteria that were infected with viruses that had radioactive proteins were
not radioactive.
 This indicates that proteins did not enter the bacteria from the viruses.
 DNA is therefore the genetic material that is passed from virus to bacteria

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Genetic material must fulfill the following criteria:
 It should be able to generate its replica (Replication).
 (ii) It should be chemically and structurally stable.
 (iii) It should provide the scope for slow changes (mutation) that are
required for evolution.
 (iv) It should be able to express itself in the form of 'Mendelian Characters’.
Comparison of DNA & RNA

Meselson and Stahl’s experiment

Meselson and Stahl performed the experiment in 1958 on E. coli to prove that
DNA replication is semiconservative.
E. coli was grown in 15NH4CI for many generations.
 15N was incorporated into newly synthesized DNA.
 This heavy DNA could be differentiated from normal DNA by
centrifugation in cesium chloride (CsCl) density gradient.
 Then they transferred these E.coli into medium with normal 14NH4Cl.
 After 20 minutes, it was found that all the DNA in the First generation
were hybrid.
 After 40 minutes, it was found that 50% DNA in the second generation
were hybrid and 50% were normal.
DNA replication
 In living cells, such as E. coli, the process of replication requires enzymes.
 The main enzyme is DNA-dependent DNA polymerase, which is used to
catalyze the polymerization of deoxynucleotides.
 In addition to DNA-dependent DNA polymerases, many additional enzymes
are required to complete the process of replication.
 For long DNA molecules, since the two strands of DNA cannot be separated
in its entire length the replication occurs within a small opening of the DNA
helix, referred to as replication fork.

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  DNA polymerase cannot initiate the polymerization itself, so a small
segment of RNA called primer is attached at replication start point.
 DNA polymerase adds nucleotides on one of the template strands called as
leading strand (the template with polarity 3‘5'). In this strand nucleotides
are added continuously and therefore it is called as continuous replication.
The direction of polymerization is 5‘3'
 On the other strand the replication is discontinuous, small fragments of DNA
are formed called Okazaki fragments which are later joined by DNA ligase.
This strand is called as lagging strand.
 Accuracy of polymerization is maintained by Proof reading and any wrong
base added is removed by DNA polymerase.

TRANSCRIPTION
The process of copying genetic information from one strand of the DNA into RNA
is termed transcription.
Compare Replication & Transcription
In replication, the total DNA of an organism gets duplicated. In transcription only a
segment of DNA and only one of the strands is copied into RNA.
Why are both the strands of DNA not copied during transcription?
 If both strands act as a template, they would code for RNA molecules with
different sequences and in turn, if they code for proteins, the sequence of
amino acids in the proteins would be different. Hence, one segment of the
DNA would be coding for two different proteins.
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 The two RNA molecules if produced simultaneously would be
complementary to each other, hence would form a double stranded RNA. This
would prevent RNA from being translated into protein.
So, if both the strands of DNA are copied during transcription, it would
complicate the genetic information transfer machinery and the exercise of
transcription would become a futile one
Explain Transcription Unit
 A transcription unit in DNA has three regions. They are (i) A Promoter (ii)
The Structural gene and (iii) A Terminator
 The enzyme DNA-dependent RNA polymerase catalyze the polymerization
in Transcription
 DNA-dependent RNA polymerase catalyze the polymerization only in one
direction, that is, 5'→3'. So the strand that has the polarity 3'→5' acts as a
template, and is also referred to as template strand.
 The other strand which has the polarity (5'→3') and the sequence same as
RNA, is displaced during transcription. It is referred to as coding strand.
 Example:
Template Strand 3'-ATGCATGCATGCATGCATGCATGC-5'
Coding Strand 5'-TACGTACGTACGTACGTACGTACG-3'
The sequence of mRNA transcribed from above DNA.
5‘-UACGUACGUACGUACGUACGUACG-3'
 The promoter and terminator flank the structural gene in a transcription
unit

 The promoter is a DNA sequence that provides binding site for RNA and is
located towards 5'-end (upstream) of the structural gene (the reference is made
with respect to the polarity of coding strand).
 The presence of a promoter in a transcription unit that also defines the
template and coding strands.

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  The terminator is located towards 3'-end (downstream) of the coding strand
and it usually defines the end of the process of transcription
 There are additional regulatory sequences that may be present further
upstream or downstream to the promoter.
Transcription Unit and the Gene
 Gene is the functional unit of inheritance, and the genes are located on
the DNA.
 The DNA sequence coding for tRNA(transfer RNA) or Rrna(ribosomal
RNA) molecule also defines a gene.
 A segment of DNA coding for a polypeptide is known as Cistron.
 The structural gene in a transcription unit is known as monocistronic in
eukaryotes or polycistronic in bacteria or prokaryotes.
 In eukaryotes, the monocistronic structural genes have interrupted coding
sequences i.e. the genes are split (split genes) or genes with coding
sequences and genes with non-coding sequences in eukaryotes.
 The coding sequences or expressed sequences are defined as exons. They
appear in mature or processed RNA.
 The exons are interrupted by introns. Introns or intervening sequences
do not appear in mature or processed RNA.
Types of RNA in Prokaryotes
 In bacteria, there are three major types of RNAs: mRNA (messenger
RNA),tRNA (transfer RNA), and rRNA (ribosomal RNA).
 All three RNAs are needed to synthesize a protein in a cell.
 The mRNA provides the template, tRNA brings amino acids and reads the
genetic code, and rRNAs play structural and catalytic role during translation.
 There is single DNA-dependent RNA polymerase that catalyzes transcription of
all types of RNA in bacteria
Process of Transcription in Prokaryotes: In prokaryotes the process of
transcription is completed in three steps:
(i)Initiation (ii) Elongation (iii) Termination
 DNA-dependent RNA polymerase catalyzes all the three steps of
transcription which are initiation, elongation, and termination.
 DNA-dependent RNA polymerase associates transiently with initiation-
factor (σ)(sigma factor) to initiate the transcription and termination- factor
(ρ) (Rho factor) to terminate the transcription.
 Initiation : RNA polymerase binds with initiation factor (sigma factor) and

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 then binds to promotor site and initiates transcription. The
binding of RNA polymerase causes local unwinding of the DNA
double helix.
 Elongation : RNA polymerase separates from sigma factor and adds
nucleoside triphosphate as substrate and polymerizes in a
template depended fashion. RNA is formed during the
process following the rule of complementary and remains bound
to enzyme RNA polymerase.
 Termination : On reaching terminator region RNA polymerase binds with
Rho factor (termination factor) As a result nascent RNA
separates. Only a short stretch of RNA remains bound to the
enzyme. Once the polymerases reach the terminator region,
the nascent RNA falls off, so also the RNA polymerase. This
results in termination of transcription.

Transcription in Eukaryotes
In eukaryotes three types of RNA polymerases are found in the nucleus (In
addition to the RNA polymerase found in the organelles) and are involved in
transcription.
RNA Polymerase I: Transcribes rRNAs
RNA Polymerase II : Transcribes hnRNA (which is precursor of mRNA).
RNA Polymerase III : Transcribes tRNA, 5 srRNA and snRNA.
 In eukaryotes the primary transcripts contain both the exons and the introns and
are non-functional

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 Hence, the primary transcript is subjected to a process called splicing where the
introns are removed, and exons are joined in a defined order
 The hnRNA (heterogeous RNA)undergo two additional processing called as
capping and tailing.
 In capping an unusual nucleotide (methyl guanosine triphosphate) is added to
the 5'-end of hnRNA
 In tailing, adenylate residues (200-300) are added at 3'-end in a template
independent manner.
 It is the fully processed hnRNA, now called mRNA, that is transported out of
the nucleus for translation.

Genetic Code
 The codon is triplet 61 codons code for amino acids and 3 codons
function as stop codons (UAG, UGA, UAA)
 (ii) One codon codes for only one amino acid, hence the codon is
unambiguous and specific.
 (iii) Some amino acids are coded by more than one codon, hence called as

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 degenerate.
 (iv) The codon is read in mRNA in a contiguous fashion. There are no
punctuations.
 (v) The code is nearly universal.
 (vi) AUG has dual functions. It codes for Methionine (met) and it also acts
as initiator codon.

tRNA, the Adapter Molecule

 tRNA occurs in the cytoplasm of the cell and it plays an important role in
protein synthesis.
 The two-dimensional structure (2-D) of a tRNA is in the form of clover leaf
 The three-dimensional structure (3-D) of a tRNA is in the form of an L-
shape
 Different types of amino acids occur in the matrix forming an ‘amino acid
pool’ in the cytoplasm.
 tRNA has an anticodon loop that has bases complementary to the code, and
it also has an amino acid accepter end to which it binds to amino acids
 tRNAs are specific for each amino acid . For initiation, there is another
specific tRNA that is referred to as initiator tRNA.
 The tRNA binds with a specific amino acid in the presence of an activating
enzyme called amino acyl-tRNA synthetase.
 The specific amino acid is attached to the 3’ end of the specific tRNA.

Translation
 Translation refers to the process of polymerization of amino acids to

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 form a polypeptide(Protein).
 The order and sequence of amino acids are defined by the sequence of bases
in the mRNA.
 A translational unit in mRNA is the sequence of RNA that is flanked by the
start codon (AUG) and the stop codon and codes for a polypeptide.
 An mRNA also has some additional sequences that are not translated and are
referred to as untranslated regions (UTR). The UTRs are present at both 5'
-end (before start codon) and at 3' -end (after stop codon). They are required
for efficient translation process.
 First step is charging of tRNA or aminoacylation of tRNA-here amino
acids are activated in the presence of ATP and linked to specific tRNA
 The cellular factory responsible for synthesising proteins is the ribosome
The ribosome consists of structural RNAs and about 80 different proteins.
In its inactive state, it exists as two subunits; a large subunit and a small
subunit.
 Protein synthesis involves three steps. They are initiation, elongation and
termination.
(i) Initiation: The mRNA binds with the small subunit of ribosome to
initiate the process of protein synthesis. Ribosome binds to mRNA at the
start codon (AUG) that is recognized by the initiator tRNA
(ii) Elongation: There are two sites in the large subunit, for subsequent
amino acids to bind to and thus, be close enough to each other for the
formation of a peptide bond. The ribosome also acts as a catalyst for the
formation of peptide bond
Complexes composed of an amino acid linked to tRNA sequentially bind
to the appropriate codon in mRNA by forming complementary base pairs
with tRNA codon. The ribosomes move from codon to codon along with
mRNA. Amino acids are added one by one and translated into
polypeptide sequences dictated by DNA and represented by mRNA
(iii) Termination: At the end, a release factor binds to the stop codon (UAA,
UAG,UGA) terminating translation and releasing the complete
polypeptide from the ribosome.

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 TRANSLATION

The Regulation of Gene expression in Eukaryotes:-

The regulation includes the following levels:-
1. Transcriptional level (formation of primary transcript)
2. Processing level (regulation of splicing)
3. Transport of mRNA from nucleus to the cytoplasm
4. Translational level.
The Regulation of Gene expression in Prokaryotes:-
Francois Jacob and a Jacque Monod proposed a mechanism for gene expression
in E. Coli, a prokaryotic bacterium, which explains the gene expression in
prokaryotes.
 The gene expression in prokaryotes is explained by ‘Operon Concept’
 The Operon Concept states that “each metabolic reaction is controlled by a
set of genes”.
 A set of genes regulating a metabolic pathway is called an Operon.
.g. lac operon, trp operon, ara operon, his operon, val operon
Lac operon in E. coli:
The operon controlling lactose metabolism. It was first elucidated by Francois
Jacob and Jacque Monod (1961).
It consists of –
a) Three structural genes: The part of DNA which transcribe mRNA for
polypeptide synthesis.

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 z gene: Codes for - galactosidase (lactose  - gal gal + glu).
 y gene: Codes for permease (increase permeability of the cell to lactose).
 a gene: Codes for a transacetylase.
b) Promoter gene (P): It is the site of attachment of RNA polymerase and initiates
transcription.
c) Operator gene (O): It is the DNA segment which controls the structural gene
when the repressor binds. It lies in between promoter and the structural gene.
d) A regulatory or inhibitor (i) gene: Codes for the repressor protein.
e) Inducer (here, lactose): it regulates switching on and off of the operon.

Functioning of Lac operon:-

When lactose (inducer) is absent:
Step 1. The i-gene synthesizes mRNA produces repressor protein
Step 2. Repressor binds to the operator region and blocks RNA polymerase
movement.
Step 3. Prevents the transcription of structural gene (remains switched off).

When lactose (inducer) is present:

Step 1. Lactose binds to the repressor making it inactive.
Step 2. Repressor fails to binds to the operator region
Step 3. The RNA polymerase binds with promoter gene (lac operon “switched on”)
and transcribe structural genes

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 Human Genome Project

HUMAN GENOME PROJECT

 The entire DNA in the haploid set of chromosome of an organism is called a
genome. In Human genome, DNA is packed in 23 chromosomes.
 Human Genome Project (1990-2003) is the first effort in identifying the
sequence of nucleotides and mapping of all the genes in human genome.
 HGP is Coordinated by the U.S. Department of Energy and the National
Institute of Health
GOALS OF HGP
 Identify all the 20,000-25,000 genes in human DNA.
 Determine the sequences of the 3 billion base pairs that make up human
DNA
 Store this information in databases
 Improve tools for data analysis.
 Transfer related technologies to other sectors, such as industries
 Address the ethical, legal, and social issues (ELSI) that may arise from the
project.
METHODOLOGIES
There are two major approaches.
1. Expressed Sequence Tags (ESTs)-Identifying all the genes that are expressed
asRNA.
2. Sequence Annotation -Sequencing the whole set of genome containing all
the coding & non-coding sequence, and later assigning different regions in the
sequence with functions.
Procedure: -
i. The total DNA from a cell is isolated.
ii. Converted into fragments of relatively smaller sizes
iii. Cloned in suitable host (e.g.: bacteria and yeast) using specialized vectors
(BAC and YAC) for amplification.
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iv. Fragments are sequenced using automated DNA sequencers (using
Frederick Sanger method).
v. These sequences were then arranged based on some overlapping regions
present in them.
vi. Alignment of these sequences using computer programs.
vii. These sequences were annotated and were assigned to each chromosome.
Salient Features of Human Genome s
i. Human genome contains 3164.7 million nucleotide bases.
ii. The total number of genes = 30,000
iii. Average gene consists of 3000 bases, but sizes vary. The largest known
human gene dystrophin contains 2.4 million bases.
iv.99.9 % nucleotide bases are exactly the same in all people. 0.1% makes each of
us unique.
v. Functions of over 50 % of the discovered genes are unknown.
vi. Less than 2% of the genome codes for proteins.
vii. Repeated sequences make up very large portion of the human genome.
viii. Chromosome 1 has most genes (2968), and the Y has the fewest (231).
ix. About 1.4 million locations where single-base DNA differences
(SNPs – single nucleotide polymorphism) occur in humans.
International Rice Genome Sequencing Project (IRGSP)
 Rice benefits from having the smallest genome of the major cereals, dense
genetic maps.
 The IRGSP, formally established in 1998, pooled the resources of sequencing
groups in 10 nations (Japan, Korea, UK, Taiwan, China,
Thailand, India, United States, Canada and France)
 India joined in June 2000 and chose to sequence a part of chromosome 11.
 Tools used in sequencing were :
BAC (Bacterial Artificial chromosomes)
PAC (P1-Phase derived artificial chromosomes). P1-Phase
derived artificial chromosome is a DNA construct that was derived from the
DNA of P1 bacteriophage. It is one type of vector used to clone DNA fragments
in E. coli cells.
 Estimated Cost of the project- $ 200 million.
How Sequenced

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Shotgun sequencing: This method involved the generation of short DNA
fragments that are then sequenced and linearly arranged. It enables full coverage of
the genome in a fraction of time required for the alternative BAC sequence
approach.
Salient Features of Rice Genome
 Rice is monocarpic annual plant, wind pollinated. It is with only 389 base pairs.
 The world’s first genome of a crop plant that was completely sequenced.
 2,859 genes seem to be unique to rice & other cereals.
 Repetitive DNA is estimated to constitute at least 50% of rice genome.
 The transposon content of rice genome is at least 35%.[ Transposon content is
the DNA sequences that have the ability to change their position within a
genome]
Applications
 To improve efficiency of Rice breeding.
 To improve nutritional value of rice, enhance crop yield by improving seed
quality, resistance to pests and diseases and plant hardiness.
DNA FINGERPRINTING
DNA fingerprinting is a very quick way to compare the DNA sequences of any
two individuals. DNA fingerprinting is a hybridization technique used to identify
the similarities of two individuals. It is a forensic tool to solve paternity, rape,
murder etc. Developed by Alec Jeffreys (British geneticist 1985).
Basis of DNA fingerprinting
Repetitive DNA: DNA fingerprinting involves identifying differences in some
specific regions in DNA sequence called as repetitive DNA, because in these
sequences, a small stretch of DNA is repeated many times. Number of repeats is
specific from person to person except in the case of monozygotic (identical) twins.
Satellite DNA: These are highly-repeated short sequences in the repetitive DNA.
Satellite DNA can be classified on the basis of following:-
a. Base composition (A:T rich or G:C rich)
b. Length of segment
c. Number of repetitive units.
These sequences normally do not code for any proteins and show high degree of
Polymorphism (variation at genetic level arises due to mutations) which form the
basis of DNA fingerprinting
Important types of Satellite DNA
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A. Micro-satellites/ SSR(Simple Sequence Repeat)- 5-8 bp long
B. Mini-satellites- 11-60 bp long, e.g.: VNTR (Variable Number of Tandem
Repeats)VNTR shows high degree of polymorphism. So, they are used as basis of
DNA fingerprinting.
Principle of DNA Fingerprinting: Short nucleotide repeats in the DNA are very
specific in each individual and vary in number from person to person but are
inherited. These are Variable Number Tandem Repeats. (VNTRs.) The VNTR
belongs to a class of satellite DNA referred to as mini-satellite. A small DNA
sequence is arranged tandemly in many copy numbers. The copy number varies
from chromosome to chromosome in an individual. Each individual inherits these
repeats from his/her parents which is used as genetic markers. One half of VNTR
alleles of the child resembles that of mother and other half the father.
Procedure in DNA Fingerprinting
In DNA fingerprinting, DNA molecules are identified by a modern technic called
Southern Blotting. Southern Blotting is a nucleic acid hybridization technique
used to identify DNA fragments with the help of DNA probe. DNA probe is a
radioactive labelled single –stranded DNA
Steps of DNA fingerprinting (Southern Blotting Technique)
(i) Isolation of DNA
(ii) Digestion of DNA by restriction endonucleases
(iii)Separation of DNA fragments by electrophoresis,
(iv) Transferring (blotting) of separated DNA fragments to synthetic membranes,
such as nitrocellulose or nylon,
(v) Hybridisation using labelled VNTR probe
(vi) Detection of hybridised DNA fragments by autoradiography
(i) Isolation of DNA
In Southern Blotting, a complete DNA molecule is isolated from a hair root or a
drop of blood or semen or from any part of the body
(ii) Digestion of DNA by restriction endonucleases
The isolated DNA is subjected to the action of restriction endonuclease enzyme.
As a result the DNA molecules become fragmented

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(iii) Separation of DNA fragments by electrophoresis
The DNA fragments are then subjected to gel electrophoresis. During this process,
the double stranded DNA fragments (ds DNA) migrate in the gel and become
separated.

(iv) Transferring (blotting) of separated DNA fragments to synthetic

membranes, such as nitrocellulose or nylon
The double stranded DNA fragments (ds DNA) separated by electrophoresis are
now treated with alkali (NaOH solution). During this process, the double stranded
DNA fragments (ds DNA) become single stranded (ss DNA) and denatured.

The single stranded denatured fragments are then transferred to a nitrocellulose

filter paper by blotting and then baked at 800 C for two hours. During this
process the single stranded denatured fragments gets fixed permanently on the
nitrocellulose filter. This process is called blotting.
(v) Hybridisation using labelled VNTR probe
The nitrocellulose filter is now placed in a solution of artificially synthesized,
radioactively labelled, denatured single-stranded DNA (ss DNA) called DNA

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probe (VNTR probe). The DNA probe binds to the complementary denatured
single-stranded DNA in the nitrocellulose filter. Thus the DNA probe hybridizes
with the DNA fragments on the nitrocellulose filter to form DNA-DNA hybrid.

(vi) Detection of hybridized DNA fragments by autoradiography

The nitrocellulose filter paper is washed to remove unbound DNA probes from the
filter. It is then photographed on to an X-ray film by autoradiography. The X-ray
image of the hybrid DNA is called DNA finger print. This DNA finger print can
be analyzed and identified.

Applications of DNA Fingerprinting

1. Identify criminals if their DNA from blood, hair follicle, skin, bone, saliva,
Sperm etc is available in forensic labs
2. It is a forensic tool to solve paternity, rape, murder etc
3. Verify whether a hopeful immigrant is, as he or he claims, really close
relative of an already established resident.
4. Identity racial groups to rewrite biological evolution.
5. It is used to study the evolutionary distance existing between two adjacent
groups.
6. It is used to diagnose many pathogens. Eg: Vibrio cholera, Neisseria
gonorrheae.

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