Chapter 6 (Molecular basis of inheritance)

1.Over the years after Mendel, the nature of the genetic material was investigated, resulting in the
realisation that DNA is the genetic material in majority of organisms.
2.Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are the two types of nucleic acid
found in living systems. Nucleic acids are polymers of nucleotide
3DNA acts as a genetic material in most organisms, whereas RNA acts as a genetic material in
some viruses.
4.RNA mostly functions as messenger. RNA has other functions as adapter, structural or as a
catalytic molecule.
5.Structure of Polynucleotide Chain
(i)A nucleotide has three parts, i.e. a nitrogenous base, a pentose sugar (deoxyribose in DNA and
ribose in RNA) and a phosphate group.
(ii)Nitrogenous bases are purines, i.e. adenine, guanine and pyrimidines, i.e. cytosine, uracil and
thymine.
(iii)Cytosine is common for both DNA and RNA and thymine is present in DNA. Uracil is present
in RNA at the place of thymine.
(iv)A nitrogenous base is linked to the pentose sugar through a N-glycosidic linkage to form a
nucleoside, i.e. adenosine and guanosine, etc.
(v)When a phosphate group is linked to 5′ —OH of a nucleoside through phosphodiester linkage, a
corresponding nucleotide is formed.
(vi)Two nucleotides are linked through 3′ -> 5′ phosphodiester linkage to form a dinucleotide.
(vii)Several nucleotides can be joined to form a polynucleotide chain.
(viii) The backbone in a polynucleotide chain is formed due to sugar and phosphates.
(ix)The nitrogenous bases linked to sugar moiety project from the backbone.
(x)The base pairs are complementary to each other.
6.In case of RNA, every nucleotide residue has an additional—OH group present at 2-position in
the ribose. Also, the uracil is found at the place of thymine (5-methyl uracil)

7.Discoveries Related to Structure of DNA

(i)Friedrich Meischer in 1869, first identified DNA as an acidic substance present in the nucleus
and named it as ‘nuclein’.
(ii)James Watson and Francis Crick, proposed a very simple double helix model for the structure
of DNA in 1953 based on X-ray diffraction data.
(iii)Erwin Chargaff proposed that for a double-stranded DNA, the ratios between adenine and
thymine and guanine and cytosine are constant and equals to one.
8.Salient Features of Double-helix Structure of DNA
(i)DNA is a long polymer of deoxyribonucleotides. It is made up of two polynucleotide chains,
where the backbone is constituted by sugar-phosphate and the bases project inside.
(ii)The two chains have anti-parallel polarity, i.e. 5′—- > 3′ for one, 3′– > 5′ for another.
(iii)The bases in two strands are paired through hydrogen bond (H—bonds) forming base pairs
(bp). Adenine forms two hydrogen bonds with thymine from opposite strand and vice-versa.
Guanine bonds with cytosine by three H—bonds. Due to this, purine always comes opposite to a
pyrimidine. This forms a uniform distance between the two strands.
(iv)The two chains are coiled in a right-handed fashion. The pitch of the helix is 3.4 nm and there
are roughly 10 bp in each turn. Due to this, the distance between a base pair in a helix is about 0.34
nm.
(v)The plane of one base pair stacks over the other in double helix. This confers stability to the
helical structure in addition to H—bonds.
9.The length of a DNA double helix is about 2.2 meters
Therefore, it needs special packaging in a cell.
(i)In prokaryotic cells (which do not have a defined nucleus), such as E.coli, DNA (being
negatively charged) is held with some proteins (that have positive charges) in a region called as
nucleoid. The DNA in nucleoid is organised in large loops held by proteins.
(ii)In eukaryotes, there is a set of positively charged proteins called histones that are rich in basic
amino acid residues, lysines and arginines (both positive).Histones are organised to form a unit of
eight molecules called histone octamer. The negatively charged DNA is wrapped around the
positively charged histone octamer to form a structure called nucleosome.
(iii)A typical nucleosome contains 200 bp of DNA helix.Nucleosomes constitute the repeating unit
of a structure in nucleus called chromatin (thread-like stained structure). Under electron
microscope, the nucleosomes in chromatin can be seen as beads-on-string. This structure in
chromatin is packaged to form chromatin fibres that further coils and condense to form
chromosomes at metaphase stage.
(iv)The packaging of chromatin at higher level requires additional set of proteins which are
collectively called Non-Histone Chromosomal (NHC) proteins.
(v)In a nucleus, some regions of chromatin are loosely packed (stains light) called euchromatin
(transcriptionally active chromatin). In some regions, chromatin is densely packed (stains dark)
called heterochromatin (inactive chromatin).

10.Transfonning Principle
(i)Frederick Griffith (1928) carried out a series of experiments with Streptococcus pneumoniae
(bacterium causing pneumonia).
(ii)According to him, when the bacteria are grown on a culture plate, some produce smooth shiny
colonies (S), while others produce rough (R) colonies.
(iii)This is because the S-strain bacteria have a mucous (polysaccharide) coat, while R-strain does
not.
(iv)Mice infected with S-strain (virulent) die from pneumonia but mice infected with R-strain do
not develop pneumonia.
(v)Griffith killed bacteria by heating and observed that heat-killed S-strain bacteria injected into
mice did not kill them. On injecting mixture of heat-killed S and live R bacteria, the mice died. He
recovered living S-bacteria from dead mice.

(vi)From this experiment, he concluded that the ‘R-strain bacteria’ had been transformed by the
heat-killed S-strain bacteria. Some transforming principle transferred from heat-killed S-strain, had
enabled the R-strain to synthesise a smooth polysaccharide coat and become virulent. This must be
due to transfer of the genetic material.
(vi)From this experiment, he concluded that the ‘R-strain bacteria’ had been transformed by the
heat-killed S-strain bacteria. Some transforming principle transferred from heat-killed S-strain, had
enabled the R-strain to synthesise a smooth polysaccharide coat and become virulent. This must be
due to transfer of the genetic material.

(v)Radioactive phages were allowed to attach to coli bacteria. As the infection proceeded, 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.
(vi) 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.
(vii)Bacteria that were infected with viruses that had radioactive proteins were not radioactive.
This indicated that the proteins did not enter the bacteria from viruses. It proved that DNA is a
genetic material that is passed from virus to bacteria.
13.Properties of Genetic Material
(i)It became establised that DNA is the genetic material from the Hershey-Chase experiment.
 (ii)In some viruses, RNA was also reported as genetic material, e.g. Tobacco mosaic viruses, QB
bacteriophage, etc.
(iii)Characteristics of a Genetic Material

(iv)It should be able to replicate.

(v)It should be chemically and structurally stable.

(vi)It should provide scope for slow changes (mutation) that are required for evolution.

(vii)It should be able to express itself in the form of ‘Mendelian characters’.

(iv) According to the above mentioned rules, both the nucleic acids (DNA and RNA) have the
ability to direct duplications.Stability can be explained in DNA as the two strands being
complementary if separated by heating come together in appropriate conditions.
(v)The 2′ — OH group present at every nucleotide in RNA is a reactive group and makes RNA
labile and easily degradable, hence it is reactive.
(vi)DNA is chemically less reactive and structurally more stable when compared to RNA. Thymine
also confers additional stability to DNA. So, among the two nucleic acids, the DNA is a
predominant genetic material.
(vii)Both RNA and DNA are able to mutate. Viruses having RNA genome and having shorter life
span mutate and evolve faster.
(viii) DNA is dependent on RNA for protein synthesis, while RNA can directly code for it. The
protein synthesising machinery has evolved around RNA. This concluded that the DNA being
more stable is suitable for storage of genetic information, while for the transmission of genetic
information, RNA is suitable.
 14.Francis Crick proposed the central dogma in molecular biology, which states that the genetic
information flows from

15.Replication Scheme for replication of DNA termed as semiconservative DNA

replication was proposed by Watson and Crick (1953). According to it,
(i)The two strands would separate and act as a template for the synthesis of new complementary
strands. .
(ii)After replication, each DNA molecule would have one parental and one newly synthesised
strand.
16.Experimental proof that DNA replicates semiconservatively, comes first from E. coli and later
from higher organisms, such as plants and human cells.
Matthew Meselson and Franklin Stahl performed the following experiments to prove this in 1958.
(i)coli was grown in a medium containing 15NH4C1 as the only nitrogen source for many
generations. 15N got incorporated into newly synthesised DNA (and other nitrogen containing
compounds). This heavy DNA molecule could be distinguished from the normal DNA by
centrifugation in a cesium chloride (CsCl) density gradient.
(ii)They then transferred the cells into a medium with normal 14NH4C1 and took samples at various
definite intervals as the cells multiplied and extracted the DNA that remained as double stranded
helices. DNA samples were separated independently on CsCl gradients to measure DNA densities.
(iii)The DNA that was extracted from the culture, one generation (after 20 min) after the transfer
from 15 N to 14N medium had a hybrid or intermediate density. DNA extracted from the culture
after another generation (after 40 min) was composed of equal amounts of this hybrid DNA and of
light DNA.
(iv)Very similar experiments were carried out by Taylor and Colleagues on Vicia faba (faba beans)
using radioactive thymidine and the same results, i.e. DNA replicates semiconservatively, were
obtained as in earlier experiments.

17.DNA replication machinery and enzymes process of replication requires a set of catalysts
(enzymes).
(i)The main enzyme is DNA-dependent DNA polymerase, since it uses a DNA template to catalyse
the polymerisation of deoxynucleotides. The average rate of polymerisation by these enzymes is
approximately 2000 bp/second.
(ii)These polymerases has to catalyse the reaction with high degree of accuracy because any
mistake during replication would result into mutations.
(iii) DNA polymerisation is an energy demanding process, so deoxyribonucleoside triphosphates
serve dual purposes, i.e. act as substrates and provide energy for polymerisation reaction.
(iv)Many additional enzymes are also required in addition to DNA-dependent DNA polymerase.
(v)(a) Replication in DNA strand occurs within a small opening of the DNA helix, known
as replication fork.
(b) DNA-dependent DNA polymerases catalyse polymerisation only in one direction, i.e. 5′ -> 3. It
creates additional complications at the replicating fork. Consequently, on one strand (template 3′-
5′), the replication is continuous, while on the other strand (template 5′-3′), it is discontinuous. The
discontinuously synthesised fragments called Okazaki fragments are later joined by DNA ligase.

18.Origin of Replication
(i)DNA polymerases cannot initiate the process of replication on their own. Also, replication does
not initiate randomly at any place in DNA. So, there is a definite region in coli DNA where the
replication originates. The region is termed as origin of replication.
 (ii)Due to this requirement, a piece of DNA, if needed to be propagated during recombinant DNA
procedures, requires a vector. The vectors provide the origin of replication.
19.RNA world RNA was the first genetic material. There are evidences to prove that essential life
processes, such as metabolism, translation, splicing, etc., have evolved around RNA.
(i)There are some important biochemical reactions in living systems that are catalysed by RNA
catalysts and not by protein enzyms.
(ii) DNA has evolved from RNA with chemical modifications that make it more stable because
RNA being a catalyst was reactive and hence, unstable.
20. There are following three types of RNAs:
(i) mRNA (messenger RNA) provides the template for transcription.
(ii) tRNA (transfer RNA) brings amino acids and reads the genetic code.
(iii) rRNA (ribosomal RNA) plays structural and catalytic role during translation.
All the three RNAs are needed to synthesise a protein in a cell.
21.Trascription is the process of copying genetic information from one strand of the i into RNA.
The principle of complementarity governs the process of transcription, except the adenosine now
forms base pair with uracil instead of l thymine.
(i) In transcription, only a segment of DNA is duplicated and on Iv one of the strands is . copied
into RNA. Both the strands are not copied because

 If both the strands code for RNA, two different RNA molecules and two different proteins would
be formed, hence complicating the genetic information transfer machinery.
 Since two RNA produced would be complementary to each other, they would form a double-
stranded RNA without translation, making the process of transcription futile.
(ii)A transcription unit in DNA is defined by three regions in the DNA which are as follows:
(a)A promoter (b) The structural gene (c) A terminator
(iii) The two strands of DNA have opposite polarity and the DNA-dependent RNA polymerase
also catalyse the polymerisation in only one direction that is 5′ -> 3′.
(iv)The strand that has the polarity 3′ -> 5′ acts as a template and is referred to as template strand.
The other strand which has the polarity (5′ -> 3′) and the sequence same as RNA (T at the place of
U) is displaced during transcription. This strand is called as coding strand.

(v)The promoter and terminator flank the structural gene in a transcription unit.
(vi)The promoter is located towards 5′ end (upstream) of the structural gene.
(vii)It is the DNA sequence that provides binding site for RNA polymerase and the presence of
promoter defines the template and coding strands. By switching its position with terminator, the
definition of coding and template strands could be reversed.
(viii) The terminator is located towards 3f-end (downstream) of the coding strand and it usually
defines the end of the process of transcription.
(ix) There are additional regulatory sequences that may be present further upstream or downstream
to the promoter.
Transcription Unit and the Gene
(i)A gene can be defined as the functional unit of inheritance.
(ii)A cistron is a segment of DNA coding for a polypeptide.
(iii) The structural gene in a transcription unit could be said as monodstronic (mostly in
eukaryotes) or polycistronic (mostly in bacteria or prokaryotes).
(iv)The coding sequences or expressed sequences are defined as exons. Exons appear in mature or
processed RNA. The exons are interrupted by introns.
(v)Introns or intervening sequences do not appear in mature or processed RNA.
(vi)Sometimes, the regulatory sequences are loosely defined as regulatory genes ,even
though these sequences do not code for any RNA or protein.
22.Transcription in prokaryotes occur in the following steps:
(i)A single DNA-dependent RNA polymerase catalyses the transcription of all types of RNA in
bacteria.
(ii)RNA polymerase binds to promotor and initiates transcription (initiation).
(iii) It uses nucleoside triphosphates as substrate and polymerises in a template depended fashion
following the rule of complementarity.
(iv) It also facilitates opening of the helix and continues elongation.
(v) Once the polymerase reaches the terminator region, the nascent RNA falls off, so also the RNA
polymerase. This results in termination of transcription.
(vi) RNA polymerase is only capable of catalysing the process of elongation.
It associates transiently with initiation-factor (a) and terminator factor (p), to initiate and terminate
the transcription, respectively. Thus, catalysing all the three steps.
(vii) Since, the mRNA does not require any processing to become active and also
since transcription and translation take place in the same compartment, many times the translation
can begin much before the mRNA is fully transcribed. As a result,transcription and translation can
be coupled in bacteria.
23. Transcription in eukaryotes have additional complexities than prokaryotes.
(i) There are at least three RNA polymerases in the nucleus other than the RNA polymerase in
organelles. The RNA polymerase I transcribes rRNAs (28S, 18S and5.8S). RNA polymerase III is
responsible for transcription of fRNA, 5srRNA and SnRNAs (small nuclear RNAs). RNA
polymerase II transcribes precursor of mRNA, the heterogenous nuclear RNA (hnRNA).
(ii) Another complexity is that, the primary transcripts contain both the exons and the ‘ introns and
are non-functional. Hence, subject to a process called splicing. In this process, introns are removed
and exons are joined in a definite order.
(iii) hnRNA undergoes additional processing called as capping and tailing. In capping, an unusual
nucleotide is added to the 5′-end of hnRNA. In tailing, adenylate residues (200-300) are added at
3’-end in a template. It is the fully processed hnRNA, now called mRNA, that is transported out of
the nucleus for translation process

Significance of these complexities are:

(i)The split gene arrangements represent an ancient feature of genome.
(ii)The presence of introns is reminescent of antiquity.
(iii) The process of splicing represents the dominance of RNA world.
1.Genetic code is the relationship between the sequence of nucleotides on mRNA and the
sequence of amino acids in the polypeptide.
2.Deciphering the Code
(i)George Gamow a physicist suggested that the genetic code should be made up of three
nucleotides. He stated that since there are only four bases and if they have to code for 20
amino acids, the code shoul d constitute a combination of bases. But, a permuta tion
combination of 43(4x 4×4) would generate 64 codons, generating many more codons than
required
(ii)Har Gobind Khorana could synthesise RNA molecules with defined combinations of
bases (homopolymers and copolymers).
(iii) Marshall Nirenberg made cell-free system for protein synthesis and finally the code
was deciphered.
(iv)Severo Ochoa enzyme (polynucleotide phosphoryiase) was also helpful in polymerising
RNA with defined sequences in a template independent manner (enzyme synthesis of
RNA).
(v)All of these investigations, finally helped to make a checker board for genetic code as
given below:

3.The salient features of genetic code are:

(i)It is a triplet, out of 64 codons 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.
(iii)Some amino acids are coded by more than one codon, hence the code is degenerate.
(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 are found in mitochondrial codons and in some
protozoans.
(vi) AUG has dual functions. It codes for methionine (met) and also acts as initiator codon.
4.Mutations and genetic code It also helps to study relationships between genes and
DNA.
(i)Point mutation occurs due to change in single base pair.Its example is a change of single
base pair in the gene for beta-globin chain of haemoglobin that results in the change of
amino acid residue glutamate to valine. It results in sickle-cell anaemia.
(ii)Frameshift mutation occurs where addition/insertion or deletion of one or two bases
changes the reading frame from the site of mutation, resulting in a protein with a different
set of amino acids. Insertion or deletion of three of its multiples of bases do not alter the
reading frame but one/more amino acids are coded in the protein translated.
(iii)Silent mutation occurs when a base change in a codon does not alter the amino acid
coded. This forms the genetic basis of proof that codon is a triplet and it is read in a
contiguous manner.
5.Francis Crick postulated the presence of an adapter molecule that would on one hand
read the code and on other hand would bind to specific amino acids. This molecule called
tRNA, then called sRNA (soluble RNA).
(i)tRNA has an anticodon loop that has bases complementary to the code and also has an
amino acid acceptor end, which it binds to amino acids.
(ii)tRNAs are specific for each amino acid, they are clover-shaped.

(iii)For initiation, there is specific JRNA (initiator iRNA). There are no tfRNAs for stop
codons.
6. Translation is the process of polymerisation of amino acids to form a polypeptide.
(i)The order and sequence of amino acids are defined by the sequence of bases in the
mRNA.
(ii)The amino acids are joined by a bond, which is known as a peptide bond. This process
requires energy.
(iii)The different phases of translation are: .
(a)Activation of amino acids occur in presence of ATP and link to their cognate fRNA, i.e.
charging of fRNA or aminoacylation of fRNA. If two such charged fRNAs are brought
close, the formation of peptide bond between them would occur energetically in presence
of a catalyst.
(b)Initiation of polypeptide synthesis occurs in ribosomes (cellular factory for protein
synthesis).

 Ribosome consists of structural RNAs and about 80 different proteins.

 In its inactive state, it exists as two subunits, i.e. a large and a small subunit.
 When the small subunit encounters an mRNA, the process of translation of the mRNA
to protein begins.
 There are two sites in the large subunit, i.e. the P-site and A-site for subsequent amino
acids to bind to and thus, be close enough to each other for the formation of a peptide
bond.
 The small subunit (with the fRNA) attaches to the large subunit in such a way that the
initiation codon (AUG) comes to the P-site.
  An mRNA also has some additional sequences that are not translated, referred as
untranslated regions (UTR). They are present at both 5′-end (before start codon) and
at 3′ end (after stop codon) for efficient translation process

(c)Elongation of polypeptide chain occur when a second fRNA charged with

an appropriate amino acid binds to the A-site of the ribosome

 A peptide bond (CO—NH) forms between the carboxyl group of methionine and the
amino group of the second amino acid. The reaction is catalysed by the enzyme
peptidyl transferase.
 The complexes composed of an amino acid linked to fRNA, sequentially bind to the
appropriate codon in mRNA by forming complementary base pairs with the fRNA
anticodon. ^ ‘ ’
 The ribosome movps ftShj cbdon to codon along the mRNA in . 5′-3′ direction.
Amino acids are added one by one, translated into polypeptide sequences dictated by
DNA and represented by mRNA.
(d) Termination of polypeptide synthesis occur when a release factor binds to the stop
codon. As a result, the polypeptide synthesis or elongation stops releasing the complete
polypeptide from the ribosome
7.Regulation of gene expression occurs at various levels. It results in the formation of a
polypetide
(i)In prokaryotes, gene expression is regulated by the rate of initiation of transcription.
(ii) In eukaryotes, regulation is achieved at four levels

 Transcriptional level (formation of primary transcript).

 Processing levels (regulation of splicing).
 Transport of mRNA from nucleus to the cytoplasm.
 Translational level.
(iii) Genes in a ceil are expressed to perform a particular function or a set of functions.
(iv)The metabolic, physiological or environmental conditions regulate expression of genes.
(v)The development and differentiation of embryo into adult organisms are also a result of
coordinated regulation of expression of several sets of genes.
(vi)In a transcriptional unit, the activity of RNA polymerase at a given promoter is in turn
regulated by interaction with accessary proteins.
(vii)The accessibility of promoter regions of prokaryotic DNA in many cases is regulated
by the interaction of proteins with sequences termed as operators,
(viii) The sequences of the operator bind a repressor protein. Each operon has its specific
operator and specific repressor. For example, lac operon interacts with lac repressor only.
8.F Jacob and J Monod were the first to describe a transcriptionally regulated system.
(i)An operon is a unit of prokaryotic gene expression which includes sequentially
regulated (structural) genes and control elements recognised by the regulatory gene
product.
(ii)The various components of an operon are:

 Structural genes Fragments of DNA which transcribe mRNA for polypeptide

synthesis.
 Promoter gene Sequence of DNA where RNA polymerase binds and initiates
transcription.
 Operator Sequence of DMA adjacent to promoter where specific repressor protein
binds.
 Regulator gene Codes for the repressor protein that binds to the operator and
suppresses its activity, hence transcription does not occur. Also, represented as *i’
gene.
 Inducer Prevents the repressor from binding to the operator. Due to this, transcription
is switched on. It may be a metabolite, hormone, etc.
9 .In lac operon. a polycistronic structural gene is regulated by a common promoter and
regulatory genes.
(i)The lac operon consists of one regulatory gene (i gene) and three structural genes (z,
yanda).
(a)i—for repressor of lac operon
z—for beta galactosidase (J3-gal) that catalyses the hydrolysis of lactose into galactose and
glucose,
y—for permease, increases the permeability of the cell, a–for transacetylase.
(b)All the three gene products in lac operon are required for the metabolism of lactose.
(ii)Lactose is a substrate for enzyme beta-galactosidase and it regulates switching on and
off of the operon, hence termed as inducer.
(iii) The lactose induces operon in the following ways

 Repressor of the operon is synthesised from the i gene.

 Repressor protein binds to the operator region of the operon and prevents RNA
polymerase from transcribing the operon.
 In presence of an inducer, such as lactose or allolactose, the repressor is inactivated by
interaction with the inducer. This allows RNA polymerase access to the promoter and
transcription proceeds.

10.Human Genome Project (HGP) was launched in the year 1990. Its total approximate
cost was 9 billion US dollars. It is a 13 year project coordinated by the US Department
of,Energy and the National Institute of Health. The project was completed in 2003. It was
closely associated with rapid development of a new area in biology called
11.The important goals of HGP are to:
(i)identify all the 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.
(iii)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
12.Methodologies of HGP is focussed on two main lines, i.e. expressed sequence tags and
sequence annotation.
(i)Expressed Sequence Tags (ESTs) method is based on identifying all the genes that are
expressed as RNA
(ii)Sequence annotation is the approach of simply sequencing the whole set of genome that
contains all the coding and non-coding sequences and later assigning different regions in
the sequence with functions.

 For sequencing, the total DNA from cell is isolated and converted in relatively smaller
sizes as fragments.
 DNA fragments are cloned in suitable host using specialised vectors, such as Bacterial
Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC).
 Fragments of DNA are then sequenced by automated DNA sequences which work on
principle developed by F Sanger.
 These sequences are arranged accordingly on the basis of overlapping regions on
DNA fragments.
 The alignments of these sequences based on computer programmes were developed.
 At last, the genetic and physical maps of the genome were constructed by collecting
information about certain repetitive DNA sequences and DNA polymorphism.
13.Salient features of human genome are as follows:
(i)The human genome contains 3164.7 million nucleotide bases.
(ii)The average gene consists of 3000 bases, but sizes vary greatly, with the largest known
human gene being dystrophin at 2.4 million bases.
(iii)The total number of genes is estimated at 30,000 much lower than previous estimates
of 80,000-1,40,000 genes. Almost all, (99.9%) nucleotide bases are exactly the same in all
people.
(iv)The functions are unknown for over 50% of the discovered genes.
(v)Less than 2% of the genome codes for proteins.
(vi)Repeated sequences make up very large portion of the human genome.
(vii)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 shed light on chromosome structure, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968) and the Y has the fewest (231).
(ix) Scientists have identified about 1.4 million locations, where single base DNA
differences, Single Nucleotide Polymorphism (SNPs) occur in humans.
14.Applications of HGP

 Knowledge from DNA sequences will define research leading to our knowledge of
biological system.
  It will enable a radically new approach to biological research.
 It can be used to study genes in a genome.
 It can create new ways to diagnose, treat and prevent the disorders that affect humans.
15.DNA fingerprinting is a quick way to compare the DNA sequences of any
two individuals.
(i)Alec Jeffreys developed the technique of DNA fingerprinting in an attempt to identify
DNA marker for the inherited diseases.
(ii)DNA fingerprinting involves identifying differences in some specific regions in DNA
sequence called as repetitive DNA (a small stretch of DNA is repeated many time).
(iii)The repetitive DNAs are separated from bulk genomic DNA as different peaks during
density gradient centrifugation. The bulk DNA forms a major peak and the other small
peaks are referred to as satellite DNA.
(iv)Satellite DNA can be classified as microsatellites, minisatellites, etc., depending on
base composition, the length of segment and number of repetitive units.
(v)These sequences show high degree of polymorphism and form the basis of DNA
fingerprinting.
(vi)Since DNA from every tissue (as blood, hair, etc) from an individual differs, they
become very useful tool in forensic applications.
(vii)As the polymorphisms are inheritable, DNA fingerprinting is the basis of paternity
testing.
(viii)Polymorphism (variation at genetic level) arises due to mutations. If an inheritable
mutation is observed in a population of high frequency, it is called DNA polymorphism.
(ix)The mutations keep on accumulating generation after generation and form one of the
basis of variability/polymorphism.
(x)There is a variety of different types of polymorphism ranging from single nucleotide
change to very large scale changes.
16.Methodology of DNA fingerprinting This technique involves Southern blot
hybridisation, using radiolabelled VNTR as a probe.
The methodology includes

 DNA is isolated and digested by the restriction endonucleases.

 DNA fragments are separated by electrophoresis.
 Separated DNA fragments are transferred to synthetic membranes like nitrocellulose
or nylon.
 Hybridisation using labelled VNTR probe.
 Hybridised DNA fragments are detected by autoradiography.
The sensitivity has been increased by use of Polymerase Chain Reaction (PCR).
17.Variable Number of Tandem Repeats (VNTRs) belongs to a class of satellite DNA
called as minisatellite.
(i) The chromosome number repeats show very high degree of polymorphism. Due to this,
the size of VNTR varies in size from 0.1-20 kb.
(ii)This DNA differs in individual to individual except in case of monozygotic (identical)
twins.
(iii)Consequently, after hybridisation with VNTR probe, the autoradiogram gives many
bands of different sizes. These bands give characteristic pattern of an individual DNA.
18.Applications of DNA fingerprinting
(i)It is useful as identification tool in forensic applications.
(ii)It is the basis of paternity testing in case of disputes.
(iii)It is used in determining population and genetic diversities and also in evolutionary biology.