Molecular Basis Of

Inheritance
 2 types of nucleic acid-
1. DeoxyRibonucleic Acid (DNA) –
Genetic material in most organisms

2. Ribonucleic Acid (RNA)-

 Genetic material in some viruses
 Messenger molecule
 Adapter molecule
 Catalytic (Ribozyme)
 Structural component
 DNA
• DNA is a long polymer of deoxyribonucleotides

• The length of DNA is usually defined as number

of nucleotides (or a pair of nucleotide referred to
as base pairs , bp) present in it. This also is the
characteristic of an organism.
i) Bacteriophage φ 174 -5386 nucleotides – ssDNA

ii). Bacteriophage lambda - 48502 base pairs (bp) -

dsDNA

iii). Escherichia coli - 4.6 × 106 bp- dsDNA

iv). Haploid content (Genome) of human DNA –

3.3 × 109 bp. - dsDNA
PACKAGING OF DNA HELIX
Distance between 2 bps=0.34 nm
(0.34X 10-9 m)

No. of bps in human= 6.6 × 109 bp

Length of human DNA = 2.2 meter

Size of human nucleus = 10-6 meter

No. Of bp in Escherichia coli =
4.6 × 106 bp

Distance between 2 bps=0.34 nm

(0.34X 10-9 m)

Length of E.coli DNA = 1.3 X 10-3 meter

or 1.3 mm

Size of cell (E.coli) = 1 micron

THE SEARCH FOR GENETIC MATERIAL
 Transforming Principle by Griffith

In 1928, Frederick Griffith, in a series of

experiments with Streptococcus
pneumoniae (bacterium responsible for
pneumonia), witnessed a miraculous
transformation in the bacteria , Host-Mice
Biochemical Characterization of
Transforming Principle :

Prior to work of Oswald Avery, Colin MacLeod

and Maclyn McCarty (1933-44), genetic
material was thought to be a protein.

They worked to determine biochemical nature of

‘transforming principle’ in Griffith's experiment.
They also discovered that protein-digesting
enzymes (proteases) and RNA-digesting
enzymes (RNases) did not affect transformation,
so transforming substance was NOT a protein or
RNA.

Digestion with DNAse did inhibit

transformation, suggesting that DNA caused
transformation. They concluded that DNA is
the hereditary material.
 TRANSDUCTION EXPERIMENT
 The unequivocal proof that DNA is the genetic material
came from experiments of Alfred Hershey and Martha
Chase (1952)
 They worked with viruses that infect bacteria called
bacteriophages (Experimental Model)
 Host – E.coli
DNA – P + , S x
Protein -P x , S +
Bacteria which was infected with viruses that had
radioactive DNA were radioactive, indicating that DNA
was material that passed from virus to bacteria.

Bacteria that were infected with viruses that had

radioactive proteins were not radioactive.

This indicates that proteins did not enter bacteria from

viruses.

DNA is therefore the genetic material that is passed

from virus to bacteria
PROPERTIES OF AS A GENETIC MATERIAL

 DNA is the predominant genetic material

 Some viruse have RNA as genetic material like

Tobacco Mosaic Virus , QB Bacteriophage etc.
 PROPERTIES OF AS A GENETIC MATERIAL
A molecule that can act as a genetic material must fulfill
following criteria:
(i) It should be able to generate its replica (Replication).
(ii) It should chemically and structurally be stable.
(iii) It should provide scope for slow changes (mutation)
that are required for evolution.
(iv) It should be able to express itself in the form of
'Mendelian Characters’.
 CENTRAL DOGMA OF MOLECULAR BIOLOGY
• Francis Crick proposed Central dogma in
molecular biology, which states that genetic
information flows from DNA → RNA → Protein.

• In some viruses , flow of information is in

reverse direction, that is, from RNA to DNA.
DNA REPLICATION
Messelson and Stahl’s Experiment (1958)
Experimental model-E.coli
1. Heavy isotope of N2- N15
2. Light isotope of N2- N14
 He used Density gradient centrifugation by
CsCl
E.coli cultured in medium containing 15NH4Cl till
entire DNA had N15
Now these cells were transferred into culture
media with 14NH4Cl
Very similar experiments involving
use of radioactive Tritiated thymidine
(H3 in place of H1)
on Vicia faba (faba beans)
by Taylor and colleagues in 1958.

The experiments proved that DNA in

chromosomes also replicate semiconservatively.
Question 1. A DNA sample containing N15 in both strands
was allowed to replicate in a medium containing N14H4Cl
for 3 generations ( Generation time = 20 min) . Calculate
number of following after 60 minutes –

1. Total no. of DNA

2. DNA molecules with N15 in both strands
3. DNA molecules with N14 in both strands
4. DNA molecules with N15 in atleast one strand
5. DNA molecules with N14 in atleast one strand
1. Total no. of DNA = 8
2. DNA molecules with N15 in both strands = 0
3. DNA molecules with N14 in both strands =6
4. DNA molecules with N15 in atleast one strand =2
5. DNA molecules with N14 in atleast one strand=8
Question 2. A DNA sample containing N15 in both strands
was allowed to replicate in a medium containing N14H4Cl
for one generation , then again transferred into N15H4Cl
for one generation , then again into N14H4Cl . Calculate
number of following after 60 minutes –

1. Total no. of DNA

2. DNA molecules with N15 in both strands
3. DNA molecules with N14 in both strands
4. DNA molecules with N15 in atleast one strand
5. DNA molecules with N14 in atleast one strand
1. Total no. of DNA = 8
2. DNA molecules with N15 in both strands = 0
3. DNA molecules with N14 in both strands =2
4. DNA molecules with N15 in atleast one strand = 6
5. DNA molecules with N14 in atleast one strand=8
Mechanism of DNA Replication - The
Machinery and Enzymes

It is enzyme mediated.

Energetically replication is a very expensive
process.
It occurs in parts due to high energy requirement.
DNA dependent DNA Polymerase
(main enzyme)

Adds 2000 bps per second

Very fast
Very accurate because Any mistake during
replication would result into mutations.

1) ORI = origin of replication

2) Activation of Nucleotides (DEEP) :
Deoxyribonucleotide,Phosphorylase Enzyme ,
Energy, two Pi required.

Deoxyribonucleoside triphosphates serve dual

purposes -
1. In addition to acting as substrates
2. they provide energy for polymerisation
reaction (two terminal phosphates in a
deoxynucleoside triphosphates are high-energy
phosphates, same as in case of ATP).
dAMP→dATP
dGMP→dGTP
dCMP→dCTP
dTMP→dTTP
3) Unwinding of DNA helix :

a) Helicase – separates 2 strands of DNA , by

breaking H-bonds.This creates Y-shaped
structure called ‘Replication Fork’

b) SSBP (single stranded binding proteins)-

bind to ss of DNA (as tetramer) to prevent
renaturation of DNA i.e reformation of H-
bonds. It provides stability to single strands
c)Topoisomerase –

 It release tension due to super coiling at

opposite end of replication fork .
 It nicks , release tension &then re-seals.

 In prokaryotes , this function is

performed by DNA Gyrase.
4) Primer formation :

 DNA-P cannot initiate replication on its own.

 So , RNA Primer (small sequence of ss RNA ,
complimentary to parent DNA strand) is formed at
3’ end of parent strand
 using enzyme DNA dependent RNA
Polymerase / RNA Primase
 RNA Polymerase /
RNA Primase

DNA-P III
5) Elongation of DNA strand

After primer addition , DNA-P III attaches

‘activated nucleotides’ to the primer → two
terminal pyrophosphate high energy are
removed → releasing energy → which is used
in forming phospho-di-ester bonds
DNA-P III adds about 2000 bps/second
DNA-P forms new strand in 5’ → 3’ direction
1.Continuous/Leading strand :
Synthesized continuously
Single primer required
Okazaki DNA fragments absent ; Faster formation
Formed on PARENT / Template strand 3’ → 5’
2.Lagging strand :
Synthesized discontinuously ; >1 primer required
Okazaki fragments present (joined by DNA ligase)
Slower
Formed on PARENT /
Template strand 5’→3’
Trick for DNA replication Steps
Oscar - Ori
Awards of -Activation of Nucleotides (DEEP)
US are -Uncoiling of Helix
(Helicase/ SSBP /Topoisomerase)
Prestigious -Primer formation (Primase)
Entertaining and –Elongation (DNA-P III)
Popular -Proof reading (DNA-P I)
Trick for DNA replication Steps
Husband - Helicase
mr. Singh - SSBP
Thought to -Topoisomerase
Propose -Primase
Dipika padukone in-DNA-P III
(DNA-P I Primer removal & Proof read)
Las vagas -Ligase (DNA)
Character Ribosomal-RNA Messenger-RNA Transfer-RNA
1.Amount in 80% (max) 5%(min) 15-20%
cell
2.Size Medium Longest Smallest
3.Mol wt Medium Maximum minimum
4.Synthesis Nucleolous (except Nucleoplasm Nucleoplasm
5s in eukaryotes-
Nucleoplasm)
5.Coding Absent Many codes 1 Anticodon/Nodoc
region
Character Ribosomal-RNA Messenger-RNA Transfer-RNA
6.Enzyme for RNA-P I RNA-P II RNA-P III
synthesis ( 5S rRNA)

7.Other names Informational/ Soluble RNA/Adapter

Nuclear/Template RNA
RNA
8.Types 3 0r 4 Many based on gene > 60 types
number
9.Function Structural , Codes for polypeptide Reads codon and
Catalytic in Brings amino acids
protein synthesis
 tRNA– the Adapter Molecule
The tRNA is also called sRNA (soluble RNA).
 Some important terms :
1.Gene :
a segment of DNA which codes for protein , t-RNA , r-RNA
It is functional unit of inheritance

2.Cistron – a segment of DNA which codes for a

specific polypeptide
Monocistronic m-RNA in Eukaryotes
(1 cistron codes for only one protein)
Polycistronic m-RNA in Prokaryotes
(1 cistron codes for > 1 protein)
 TRANSCRIPTION
The process of copying genetic information
from one strand of DNA into RNA

DNA dependent RNA Polymerase

Like DNA replication , The principle of
complementarity governs the process of
transcription,

except the adenosine , now forms base

pair with uracil instead of thymine

Unlike DNA replication , Only One

strand of DNA forms RNA
Why both the strands are not copied during
transcription?
Template/Non-coding/Anti-sense strand of DNA
Strand with polarity - 3’→ 5’
RNA is synthesized on this strand in 5’→3’as
RNA Polymerase works in 5’→ 3’
Trick-TAN

 Coding /Non-template/ Sense strand of DNA

Strand with polarity - 5’→ 3’
RNA formed is similar to coding strand ,
but formed on template strand
Transcription Unit

It is segment of DNA involved in transcription.

A transcription unit in DNA is defined
primarily by the three regions in the DNA:

(i) A Promoter
(ii) The Structural gene
(iii) A Terminator
RNA polymerase
Forms RNA in 5’→ 3’ on template strand
In prokaryotes – only one type which forms all types of
RNA

In eukaryotes – 3 types of RNA Polymerase

1.RNA polymerase I (nucloelus) transcribes rRNAs
(28S, 18S, and 5.8S)
2.RNA polymerase II (nucleoplasm)transcribes
mRNA – the heterogeneous RNA (hnRNA).
3. RNA polymerase III (nucleoplasm) is transcribes
tRNA , 5s rRNA, scRNAs and snRNAs
Sigma factor (σ ) Rho factor (ρ)
Structural gene –
In eukaryotes – monocistronic genes

In eukaryotes , Split gene present =

1.Functional Exons /coding/expressed sequences
(appear in mature or processed RNA)

2.with intervening Introns (junk DNA/ intermediate

DNA/non-functional / non-coding/don’t appear in
mature or processed RNA)
 Trick - NICE
 TRANSCRIPTION IN PROKARYOTES
There is single DNA-dependent RNA polymerase
that catalyse transcription of all types of RNA in
bacteria.
It occurs in cytoplasm.

3 steps –
1. Initiation
2. Elongation
3. Termination
RNA Polymerase uses nucleoside triphosphates as
substrate and polymerises in a template depended
fashion following the rule of complementarity.

It somehow also facilitates opening of the helix and

continues elongation till it reaches terminator site

Only a short stretch of RNA remains bound to

enzyme.
 Post transcriptional modification in Eukaryotes

Capping (5’) & Tailing (3’)

 Difference between transcription in prokaryotes
and eukaryotes
PROKARYOTES EUKARYOTES
1.Occur in Cytoplasm In Nucleus
2.Only one type of RNA-P 3 types of RNA-P
3.No Post-Transcriptional -Post-Transcriptional modification
modification -Splicing
-No Splicing
4.Both transcription and Followed by Translation in
translation in cytoplasm cytoplasm
The split gene arrangements represent
probably an ancient feature of genome.

The presence of introns is reminiscent of

antiquity,

and the process of splicing represents the

dominance of RNA-world.
Ques) The sequence of Antisense strand of dsDNA
5’ AATTATGCGGCC3’
Find out sequence of-

1. Coding strand

2. Template strand

3. mRNA transcribed by it
 GENETIC CODE
Genetic code - It is a sequence of nitrogen
bases/nucleotides on m-RNA , which have coded
information for amino acid synthesis.

 Direct sequence of amino acids during

synthesis of proteins.

Codon – it is a sequence of nitrogen bases which

codes for a specific amino acid synthesis.
Codons are triplet in nature
This was a very bold proposition,
because a permutation combination of 43
(4 × 4 × 4) would generate 64 codons.
n
NO. OF CODONS = 4 , where
n = no. of nitrogen bases in each codon
Salient features of Genetic Code: NO DUCTS
(i) The codon is triplet. 61 codons code for
amino acids and 3 codons do not code for any
amino acids, hence they function as stop
codons.

(ii) One codon codes for only one amino acid,

hence, it is unambiguous and specific
(except GUG-Valine/methionine at initiation
point)
(iii) Some amino acids are coded by more than one
codon , hence the code is Degenerate.
3 aa – 6 codons (SAL-Serine , Arginine, Leucine)
5 aa – 4 codons
1 aa – 3 codons
9 aa – 2 codons
Total 18 aa show degeneracy
2 aa are coded by only 1 codon
(Exception to Degeneracy)
eg- Methionine by AUG & Tryptophan by UGG
3 Codons are Stop codons
(iv) The codon is read in mRNA in a contiguous
fashion. There are no punctuations.

(v) The code is nearly universal:

for example, from bacteria to human UUU

would code for Phenylalanine (phe).
Some Exceptions to this rule have been
found in mitochondrial codons, and in some
protozoans.
(vi) AUG has dual functions.

It codes for Methionine (met) ,

and it also act as initiator codon

(vii) Non-overlapping
Eg- AUGUUUGUG
3 Codons = 3 Amino acids
Salient features of Genetic Code:

NO – Non-Overlapping
D - Dual and Degeneracy
U - Universal
C – Commaless (no punctuations)
T - Triplet
S - Specific and unambiguous
Stop/Terminator/ Non-sense codons: 3
Do not synthesise any amino acid
1.UAA – Ochre (Ullu Aya Aya)

2.UAG – Amber(Ullu Aya Gaya)

3.UGA – Opal (Ullu Gaya Aya)

 61 codons – Sense codons , codes for 20 aa

 Initiation/Start codon –
1. AUG
2. GUG (only if at 1st position)
UCAG
 Mutations and Genetic Code
Insertion or deletion of three or its multiple bases insert
or delete one or multiple codon hence one or multiple
amino acids, and reading frame remains unaltered from
that point onwards. Such mutations are referred to as
Frame-shift insertion or deletion mutations.
1.Frame shift insertion or deletion mutation –

Insertion or deletion of one or two bases

changes reading frame from point of insertion or
deletion

Insertion or deletion of three or its multiple bases

insert or delete one or multiple codon hence one
or multiple amino acids, and reading frame
remains unaltered from that point onwards
RAM HAS RED CAP
RAM HAS BRE DCA P
RAM HAS BIR EDC AP
RAM HAS BIG RED CAP

RAM HAS EDC AP (R removed)

RAM HAS DCA P (RE removed)
RAM HAS CAP (RED removed)
This forms genetic basis of proof that codon is
a triplet & it is read in a contiguous manner.
2. Substitution / Point mutation – complete reading
of frame is not altered
 THE FAT CAT ATE THE RAT (BAT)
a)Transition –
if Pu is mutated to Pu or Py to Py
1. AUG (methionine) → GUG(valine)
2. AUG (methionine) → ACG (threonine)

b)Transversion –
if Pu is mutated to Py or Py is mutated to Pu
1. AUG (methionine) → CUG(Leucine)
2. AUG (methionine) → AAG (Lysine)
 TRANSLATION
The process of polymerisation of amino acids to form a
polypeptide.

It occurs in cytoplasm in both prokaryotes and

eukaryotes.
Cellular factory for protein synthesis is Ribosome.

The order and sequence of amino acids are defined by

sequence of bases in mRNA.
When 2 subunits of ribosome combines ,
there is formation of 3 sites

1.P site (Peptidyl site)

2.A site ( Aminoacyl site)

3.E site ( Exit site)

Process of translation :
1.Activation of amino acid :
In the first phase itself amino acids are
activated in presence of ATP

Amino acid + ATP

Aminoacyl AMP- enzyme complex + PP

AA + ATP + Aminoacyl synthetase
enzyme (E) + Mg2+
→ AA-AMP-E Complex + Ppi

• There is a separate “Amino acyl t-RNA

synthetase enzyme for each kind of
amino acid
2. Charging of tRNA or Aminoacylation of
tRNA :

Activated Amino acids linked to their cognate tRNA

AA-AMP-E Complex + specific t RNA →

tRNA –AA (Charged tRNA) + AMP + Enzyme
3. Formation of polypeptide chain :

The amino acids are joined by a bond which is

known as a peptide bond.

Formation of a peptide bond requires energy.

The Ribosome also acts as a catalyst (23S

rRNA in bacteria is enzyme- Ribozyme) for
formation of peptide bond.
A translational unit in mRNA

 The sequence of RNA that is flanked

by start codon (AUG) at 5’ and stop codon at 3’

and codes for a polypeptide.

A) Binding of mRNA with smaller subunit of ribosome

B) Binding of charged tRNA with m RNA-Ribosome

complex

C) Attachment of Larger subunit of Ribosome

 m RNA- tRNA Ribosome Initiation complex
 P-site formed (where 1st charged tRNA is attached)
Amino acids are added one by one, translated
into Polypeptide sequences

dictated by DNA and represented by mRNA.

 Regulation of Gene Expression :
It is Regulation over functioning of genes i.e
mechanism at molecular level by which a gene
expresses itself into phenotype of organism.
It can be exerted at four levels (Eukaryotes) :
(i) Transcriptional level during formation of
primary transcript (hn RNA)
(ii) Processing level (regulation of splicing)
(iii) Transport of mRNAs from nucleus to
cytoplasm
(iv) Translational level
 Gene regulation in Prokaryotes:
 Operon Model :
•An operon is a segment of DNA that functions as
single regulated unit comprising
1. one or more Structural genes
( → mRNA → polypeptide )
2. a Regulator gene (r/i) – produce Repressor or
Activator protein
3. a Promoter gene (p) - bind with RNA-P
(self-expression absent)
4. an Operator gene (o) - self-expression absent
First operon lac–Operon was

Given by Geneticist , Francois Jacob and

a biochemist, Jacque Monod (1961) in
E.coli
The Lac operon
Geneticist , Francois Jacob and a biochemist,
Jacque Monod
a transcriptionally regulated system
 In lac operon (here lac referes to lactose),

a Polycistronic structural gene is regulated by a

common promoter and regulatory gene
Such arrangement is very common in bacteria ,
referred to as operon
If Lactose absent :
If Lactose present :
If lactose absent , Switch Off
If lactose present , Switch On

If lactose absent , Repressor protein

(i-gene) + Operator gene = Switch Off

If lactose present , Repressor protein

(i-gene) + Inducer (Lactose/ Allolactose)
= Switch On
Trick : LION

Lactose (Inducer)
Inducible operon
ON mode
N-Negative regulation
Human Genome project (HGP) :

HGP is a mega project

Started by U.S. Department of Energy and
National Institute of Health
For sequencing human genome
In 1990
Welcome Trust (UK) joined project as a major
partner
Later on Japan, France, Germany, China & some
other countries also joined it.
Expected scheduled completion – 2005

But completed in 2003 , in 13 years

Till 2003 , all 23 (21 A + X +Y)

chromosomes except chromosome no. 1

Last chromosome to be sequenced is

chromosome 1 (May 2006)
Why called Mega project ???

1.Human genome is said to have approximately

3 x 109 bp,

and if the cost of sequencing required is

US $ 3 per bp (the estimated cost in beginning),
total estimated cost of the project would be
approximately 9 billion US dollars
2.If obtained sequences were to be stored in
typed form in books,

and if each page of the book contained 1000

letters
and each book contained 1000 pages,
then 3300 such books would be required to
store the information of DNA sequence from a
single human cell.
3.The enormous amount of data expected to be
generated also necessitated the use of high
speed computational devices for data storage
and retrieval, and analysis.

HGP was closely associated with the

rapid development of a new area in biology
called as Bioinformatics (use of high speed
computational devices for data storage and
retrieval, and analysis)
Goals of HGP : TrICK : Good Simple SITA

(i) Identify all approximately 20,000-25,000

Genes in human DNA

(ii) Determine the Sequences of the 3 billion

chemical base pairs that make up human DNA;
(iiii) Store this information in databases;

(iv) Improve tools for data analysis;

(v) Transfer related technologies to other

sectors, such as industries;

(vi) Address the ethical, legal, and social issues

(ELSI) that may arise from the project.
Many non-human model organisms have also
been sequenced – Trick : BAD CRY

1. Bacteria
2. Yeast
3. Caenorhabditis elegans
(a free living non-pathogenic nematode)
4. Drosophila (the fruit fly)
5. Plants (Rice and Arabidopsis)
 Methodology :

Two approaches have been recognized

for analysing human genome

(i) ESTs or Expressed Sequence Tags :

 To identify all the genes that are
expressed as RNA.
(ii) Sequence annotation :

 Sequencing both coding and

noncoding regions of whole genome
and later assigning different regions
in sequence with functions

 Blind approach
For sequencing,

1.Total DNA from a cell is isolated

2.Then converted into random fragments of

relatively smaller sizes

(recall DNA is a very long polymer, and there are

technical limitations in sequencing very long
pieces of DNA)
3.Then , Cloned in suitable host using
specialised vectors.

The cloning resulted into amplification of

each piece of DNA fragment so that it
subsequently could be sequenced with ease.
Commonly used hosts- Bacteria & Yeast
Vectors-
1. BAC (Bacterial Artificial Chromosomes)
2. YAC (Yeast Artificial Chromosomes).
4.Fragments were sequenced using
Automated DNA sequencers

that worked on the principle of a method

developed by Frederick Sanger.

Sanger is also credited for developing method

for determination of amino acid sequences in
proteins.
5.These sequences were then arranged based on
some overlapping regions present in them.
This required generation of overlapping
fragments for sequencing.
Alignment of these sequences was humanly not
possible.
Therefore, Specialised Computer Based
Programs were developed . These sequences were
subsequently annotated and were assigned to
each chromosome
Another challenging task was assigning
genetic and physical maps on genome.

This was generated using information on

polymorphism of restriction endonuclease
recognition sites & some repetitive DNA
sequences known as Microsatellites

One of applications of polymorphism in

repetitive DNA sequences - DNA fingerprinting.
Salient features of Human Genome :

(i)The human genome contains 3164.7 million

nucleotide bases (3.1647 Billion)

(ii) The total number of genes is estimated at 30,000–

much lower than previous estimates of 80,000 to
1,40,000 genes. Almost all (99.9 per cent) nucleotide
bases are exactly the same in all people.
(iii) The average gene consists of 3000 bases,
but sizes vary greatly
largest known human gene being dystrophin at
2.4 million bases
Smallest human gene-TDF (Testis Determining
Factor on Y-chromosome – 14 bases)

(iv) Chromosome 1 has most genes (2968),

& Y has the fewest (231)
(v) The functions are unknown for over 50
% of discovered genes

(vi) < 2 % of genome codes for proteins

(vii) Repeated sequences make up very large
portion of the human genome.

Repetitive sequences are stretches of DNA

sequences that are repeated many times,
sometimes hundred to thousand times.
They are thought to have no direct coding
functions,
but they shed light on chromosome
structure, dynamics and evolution.
(viii) Scientists have identified about 1.4 million
locations where single- base DNA differences
(SNPs – Single Nucleotide
Polymorphism, pronounced as ‘snips’) occur in
humans

This information helps in processes of finding

chromosomal locations for disease-associated
sequences
and Tracing human history.
DNAFingerprinting
(DNA Profiling / Typing / Imprinting)
Historical Aspect :
Sir Alec jeffreys (1984) developed
DNA fingerprinting technique.

Dr.K.kashyap and Dr. Lalji Singh started the

fingerprinting technology in India
Centre for DNA Fingerprinting and Diagnosis
(CDFD) , Hyderabad
What is DNA-fingerprinting :

It is a technique to identify a person on

the basis of his//her DNA specificity

Technique of determining nucleotide

sequences in certain DNA segments , which is
unique to an individual (0.1% = 3 million bp)
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.

These repetitive DNA are separated from bulk

genomic DNA as different peaks during density
gradient centrifugation.
Bulk DNA forms a major peak and the other
small peaks are referred to as Satellite DNA.
 Short tandem/Satellite DNA – based on Base composition,
Length of segment & Number of repetitive units (Trick: BSNL)
i. Microsatellite / Short tandem repeat (STR) :
1-6 bp long
ii. Mini-satellite / VNTR : 11-60 bp long
don’t code for any proteins
Form a large portion of human genome
Show high degree of polymorphism
Form basis of DNA fingerprinting (Unique to an individual)
copy number varies from chromosome to chromosome in
an individual
Size of VNTR varies in size from 0.1 to 20 kb
VNTR is flanked on both sides by Restriction sites
Depending on Base composition (A : T rich or G:C
rich), Length of segment & Number of repetitive
units (Trick: BSNL), Satellite DNA is classified into
many categories, such as micro-satellites, mini-
satellites etc.
Since DNA from every tissue (such as blood,
hair -follicle, skin, bone, saliva, sperm etc.),
from an individual show the same degree of
polymorphism, they become very useful
identification tool in forensic applications.

Further, as polymorphisms are inheritable

from parents to children, DNA fingerprinting is
the basis of paternity testing, in case of
disputes.
 DNA Polymorphism :

Polymorphism (variation at genetic level)

Polymorphism in DNA sequence is the basis

of genetic mapping of human genome as well
as of DNA fingerprinting

Arises due to mutations

New mutations may arise in an individual either in
somatic cells or in the germ cells (cells that generate
gametes in sexually reproducing organisms).

If a germ cell mutation does not seriously impair

individual’s ability to have offspring who can transmit
the mutation, it can spread to the other members of
population (through sexual reproduction).
Allelic sequence variation has traditionally been
described as a DNA polymorphism if more than one
variant (allele) at a locus occurs in human population
with a frequency greater than 0.01.
There is a variety of different types of
polymorphisms ranging from single nucleotide
change to very large scale changes.

 For evolution and speciation, such

polymorphisms play very important role
In simple terms, if an inheritable mutation is
observed in a population at high frequency, it is
referred to as DNA polymorphism.
The probability of such variation to be observed
in non- coding DNA sequence would be higher as
mutations in these sequences may not have any
immediate effect/impact in an individual’s
reproductive ability.
These mutations keep on accumulating
generation after generation, and form one of the
basis of variability/polymorphism.
 Technique of DNA fingerprinting

(i) The DNA is isolated from the nuclei of

white blood cells or spermatozoa or the
hair follicle cells.

(ii) DNA multiplied/Amplified through PCR

technique
(iii) Restriction endonuclease enzyme
performs digestion of DNA molecules
 The former cuts DNA in to fragments
 Fragments of DNA also contain VNTRs

(iv) Gel electrophoresis is used to separate

these fragments according to their size.

Denaturation of DNA by Alkali to split them into

ssDNAs
(v) SS DNA fragments of the gel are shifted onto a
Nylon paper/Nitrocellulose membrane by
Southern blotting technique

(vi) Hybridization of VNTR on nylon membrane with

Radioactive ssDNA probes (VNTR Probe)
(viii) Detection of hybridised DNA fragments by
Autoradiography

 Now nylon membrane is exposed with X-ray and

mark places where radioactive DNA probes have
bound to the DNA fragments

 These places are marked as dark bands (DNA

Fingerprints , unique to an individual)
(i) Isolation of DNA
(ii) Amplification of DNA by PCR (if required)
(iii) Digestion of DNA by restriction endonucleases
(iv) Separation of DNA fragments by electrophoresis

(v) Transferring (blotting) of separated DNA fragments to

synthetic membranes, such as nitrocellulose or nylon

(vi) Hybridisation using labelled (Radiolabelled) VNTR probe

(vii) Detection of hybridised DNA fragments by
Autoradiography
 Technique of DNA Fingerprinting :

 Initially developed by Alec Jeffreys.

He used a Satellite DNA as probe that
shows very high degree of polymorphism.
It was called as Variable Number of Tandem
Repeats (VNTR 11-60 bp long)/Minisatellites
The technique, as used earlier, involved
Southern blot hybridisation using radiolabelled
VNTR as a probe.
Consequently, after hybridisation with VNTR
probe, the autoradiogram gives many bands of
differing sizes

These bands give a characteristic pattern for

an individual DNA

It differs from individual to individual in a

population except in the case of monozygotic
(identical) twins.
The sensitivity of the technique has been
increased by use of polymerase chain reaction

Consequently, DNA from a single cell is

enough to perform DNA fingerprinting analysis

In addition to application in forensic science,

it has much wider application, such as in
determining population and genetic diversities
 Currently, many different probes are used to
generate DNA fingerprints.