Sex Chromatin

Barr and Bertram in 1949 discovered presence of drumstick like body in nuclei of
nerve cell in female cat. Human female contain two X chromosome out of which
one X remain inactive and present as coiled structure in nuclei called as Sex
chromatin /Barr body .

In human female there are two X chromosome where as in Male there is only one
X chromosome. Out of Two X one X in somatic cell become inactive and represent
as coiled dark stained chromatin mass called Sex chromatin.

Identification of sex chromatin is an easy and quick method to determine sex. Most
of body cell of female show presence of sex chromatin. Skin, oral mucosa, Vaginal
or Urethral epithelium show presence of sex chromatin. Laboratory Investigation is
done with Buccal mucosal smear which is obtained by scrapping of cheek. Slide
spread is made and fixed in Alcohol and stained with basic dyes. The slides are
observed under high magnification for the presence of sex chromatin or Barr body.
If the cell are chromatin positive then the sex is identified as female. Barr body is
present in nucleus as drumstick like structure. It is absent in male. For
determination of sex Barr body is not very satisfactory method. Karyotyping
determine sex accurately.

One Barr body is present in normal female. A patient of Klinefelter’s Syndrome

(XXXY) has 2 Barr bodies. A patient of Turner’s syndrome (XO) has no Barr
bodies

Significance – Presence of Barr bodies proves that somatic cell of that individual
contain 2 or more X chromosomes.

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Lyon ‘s Hypothesis
Mary F Lyon in 1962 stated hypothesis that during early embryogenesis one of
two X chromosome in females become condensed and inactive which form Barr
body. The process of X inactivation is referred as Lyonization

Key point of Lyonization

1. Out of two X chromosomes only one X become inactive

2. Inactivation of X chromosome occurs early in embryonic life at 15 to 16 th
day of gestation.
3. Inactivation of X chromosome is at random. In some cell maternal X
become inactive and in some cell paternal X become inactive
4. Once a particular X chromosome become inactive then after same X
inactivation is followed in subsequent cell division
5. Every healthy female is kind of Mosaic in respect to gene present on X
chromosome.
6. Inactive X is late replicating during Mitosis
7. Number of Barr body is always less than the number of X chromosome in
females

Genetic signification of X inactivation

1. Dosage Compensation - it justify why X linked gene product are of same

dose in female and male in spite of two x chromosome of female
2. Variability of Expression – Inactivation of X is random which provide a
variability for phenotypic variation
3. Mosaicism – females are Mosaic having two cell line

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Karyotyping
Karyotyping is procedure to obtain Karyotype of an individual. In this procedure
metaphase chromosome of somatic cell are obtained, photographed and arranged
according to standard classification.

Cell are obtained from following sources for karyotyping

• Lymphocyte from Peripheral Blood

• Fibroblast from Skin
• Bone Marrow cells
• Chronic Villi
• Amniotic Fluid cells

Step of Karyotyping

1. Collection of Blood – 5 ml of venous blood is collected in sterile condition.

Heparin is mixed to avoid clotting. Lymphocyte are separated from red
blood cells
2. Culture Media – Culture Media contain RPMI 1640 and Fetal Calf Serum
which provide nourishment to developing lymphocyte. Phytohaemagglutinin
is also added to stimulate cell division of lymphocytes. Antibiotic in culture
media prevents infection of culture.
3. Incubation- Culture vial is put in Incubator for 3 days at 37˚Cduring which
lymphocyte divide rapidly.
4. Harvesting - At the end of 72 hours Colchicin is added to culture vial.
Cholchicin prevent formation of spindle and arrest the cell division during
metaphase. In Metaphase Chromosomes are maximally condensed and
easily visible.
5. Hypotonic Saline – Lymphocyte are separated by centrifugation. Now cells
are treated with Hypotonic Saline. Hypotonic Saline cause cell to swell and
chromosomes are separated
6. Fixation – Fixation is done with a mixture of Glacial Acetic Acid and
Methanol in 1:3 proportions. Cell suspension is dropped on chilled slides
from height which rupture cell wall and chromosomes spread on slide. This
spread is referred as Metaphase Spread.

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7. - Metaphase spread is then stained and micro photographed. Individual
chromosome are cut and arranged to obtain karyotype.

Application of Karyotyping
• Congenital Malformation,
• Mental Retardation
• Repeated Abortion
• Prenatal Diagnosis

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Chromosomes Banding
After the karyotyping it is very essential to stain chromosomes for identification of
Chromosomal Anomalies. There are various technique of banding which will help
to identify pair of chromosome and structural abnormalities.

1. G –Banding--- It is Giemsa bending in which chromosome preparation are

first treated with Trypsin solution. Trypsin denatures protein and then slides
are stained with Giemsa gives Dark and Light band. This technique is called
G banding.
2. Q- Banding ---In this technique chromosomes are stained with
Quinacrine Mustard. The banding patterns are similar to G-banding but it
is observed under Ultra Fluorescent microscope.
3. R-Banding---It is a reverse banding technique in which chromosome are
Pre –Heated before staining with Giemsa that gives pattern of staining
which is reverse to G-banding .
4. C-Banding --- In this banding technique Centromere and Secondary
Constriction are stained

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Fluorescent in situ Hybridization (FISH)
This is new diagnostic technique. Principal of this technique is that a single
stranded DNA probe has ability to anneal with its complimentary target sequence
was ever located on metaphase chromosome. DNA probes are radioactively
labeled. The probe is conjugated with a fluorescent label and visualized under UV
light. The site where DNA is labeled hybridizes with its complimentary DNA
sequence on Nitrocellulose Filter, which can be localized by Autoradiography.
FISH technique is widely technique due to its fast results.

Various types of chromosome –specific probes can be used. This technique can be
for complex chromosomal arrangement such as deletion, insertion, translocations,
and ring chromosomes.

Types of FISH probes are used

• Centromere probe- used to identify specific chromosomes

• Chromosome specific unique sequence probe- used to identify

submicroscopic deletion and duplications

• Locus –specific probe- DNA sequence of homologues chromosomes and

specific gene location can be identified by locus specific prob. This is used
in tumor, prenatal, postnatal samples

• Whole chromosome paint probe(WCB)- to visualize entire length of

chromosome

• Multicolor Spectral Karyotyping (SKY)- multiple probes labeled with

different fluorescent dyes are used to paint all chromosomes simultaneously.
It helps to identify numerical chromosomal abnormality

FISH can be applied at interphase stage so it can be used to get rapid diagnosis in
investigation like Chorionic villous sample without culturing the cell.

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Chromosomal Disorder or Chromosomal Aberration
Any alteration in normal chromosomal complement is called Chromosomal
Aberration. Chromosomal disorder are classified in two ways

Type of abnormality

• Structural abnormality- involves change in structure of chromosome. A

part of chromosome is missing, duplicated or broken lead to this type of
abnormalities
Eg Deletion, Inversion, Ring chromosomes, Isochromosome, Translocation
• Numerical abnormality- involves change in number of chromosome. There
is increase or decrease in no of chromosomes .the abnormalities occur in
gametogenesis when homologous pair of chromosomes separate from each
other in meiosis I-called Non-disjunction. Eg - Trisomy 21(Down ‘s
Syndrome ),trisomy 13( Patau’s syndrome),Trisomy 18( Edward’s
syndrome),Turner syndrome (45X, Klinefelter’s syndrome (47,XXY) , XXX
females, XYY males.
• Factors responsible for chromosomal aberrations
• Ionizing radiation
• Viruses
• Chemical carcinogens
• Advanced maternal or paternal age
• Non disjunction

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Structural Chromosomal aberration –
Abnormalities in structure of chromosome lead to loss of genes, gain of genes.
Change in position of gene lead to alteration in structure of chromosome

Following are various types of structural disorders

Deletion
• Abnormal breakage occur in part of chromosome and broken part is lost as it
has no centromere
• In deletion there is loss of genetic material from genome
• If the loss is more than 2% of gene is lethal for survival
• Terminal Deletion- Single breakage of part of chromosome at terminal end
• Interstitial Deletion - Double breakage of intermediate part of
chromosome and broken part is lost

• There are two type of deletion

• Microscopic deletion-this can be only visualized microscopically by
karyotyping if deletion is quite large eg- deletion of short arem of 5
chromosome in cri-du-chat syndrome
• Submicroscopic deletion –this can not be visualized by usual karyotype
require FISH eg Paderwili (15), Angelman (15),Williams(7)

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 Inversion
• It involve single chromosome which break at two point
• Broken segment rearrange itself by inverting position
• It could be pericentric or paracentric
• No clinical problem is seen in carriers as there is no loss of genetic material
• A chromosome with Inversion give rise to abnormal gamete

Ring Chromosome
• It is rare abnormality where chromosome forms circle or Ring
• Breakage occur near tip of each arm
• Broken arm stick to each other and subsequently fuse with each other
• Two distal fragment are lost
• During cell division such ring chromosome leads to abnormal migration and
produce gametes with genetic alterations

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 Isochromosome
• Due to incorrect splitting of centromere results in formation of isochromosoe
• Splitting lead to two isochromosomes that has two genetically identical arms
• In one cell there is duplication of one entire chromosome arm and deletion
of other chromosome arm

Translocation
There is exchange of genetic material between two chromosomes. Translocation
can be balanced or unbalanced. The carrier of balanced translocation are normal
themselves, but children are affected. Two types of translocation

Reciprocal translocation

• Translocation occur between two non-homologous chromosome after

breakage and exchange of fragments
• It is balanced due to rearrangement of genetic material no phenotypic effect
is seen in carrier

Robertsonian translocation

• Translocation occur at or near centromere in two acrocentric chromosomes

• Short arm of both chromosome are lost and long arm fuse together
• About 4% cases of down’s syndrome is due to robertsonian translocation

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Numerical abnormality
Change in number of chromosomes leads to numerical abnormality
Polyploidy- multiples of haploid number result in triploidy (3n),
tetraploidy and so on. Embryos with these types of polyploidy result in
spontaneous abortions or die soon after birth.
Aneuploidy-in this condition number of chromosome is altered one or
more. There are two types
Hyperploidy- one or more additional chromosomes to diploid number
eg. Trisomy (2n+1), tetrasomy (2n+2)
Hypoploidy- one or more number of chromosomes is less to the diploid
number eg. Monosomy (2n-1), Nullisomy (2n-2)

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Trisomy 21 /Down’s Syndrome
It is chromosomal abnormality involving trisomy of 21 chromosome. It was first
describe by Down in 1866. It occurs in 1 in 700 live birth. Incidence increase with
advancing maternal age due to risk of non-disjunction occurring in aging ovary.

Incidences – once in every 800 to 1000 live births

Cause- it occur due to failure of separation of 21st pair or chromosome during

meiosis. This result in an extra chromosome on 21st pair

Clinical Feature
• Mental Retardation with IQ level between 25 to 75
• Shot suture
• Hypotonia
• Small round head /Brachy-cephaly
• Prominent gape bet 1st and 2nd toes
• Dermatoglyphics – atd angel- greater than 57˚ , presence of Simian crease
• Head and neck- small rounded head(Brachy-cephaly), oblique palpable
fissure , Epicanthic folds, protruding tongue, small ears, slopping palpebral
fissure, Flat nasal bridge
• Hand- broad and short hand, short fingers, semian crease present in hand
• Cvs- Congenital cardiac defects, Ventricular and Septal defects are more
common
• Abdomen and pelvis- umblical hernia, dudoneal atresia, cryptorchidism
• Radiological finding- hypoplasia of midphalanx of fifth finger, Iliac index is
less than 60˚

Genotype – Down’s syndrome is due to following chromosomal abnormalities

• Trisomy
• Translocation
• Mosaicism

Life Span- Average life is 16 to 18 yrs. Most of affected adult develop Alzheimer’s
disease in later life

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Counseling

Prenatal diagnosis of Down’s syndrome can be done by Amniocentesis, Triple

marker test ( Fetoprotein, Oestriol , Chorionic Gonadotropin)done at 16 week of
gestation , Inhibin –A level in maternal serum.

Turner Syndrome(45,XO)
It is a sex chromosomal disorder having only one X chromosome. It was first
describe by Turner in 1938. It occurs once in 5000 births. Phenotype will be
female

Clinical feature

• Short suture
• Short and webbed neck
• Low posterior hair line
• Primary Amenorrhea ,Scanty pubic hair ,Flat chest, Undeveloped breast
• Sexual Infantilism
• Head and neck- Epicanthic fold, Small Mandible, High Palate, defective
vision, narrow maxilla, webbed neck,
• Cvs- Coaraction of aorta ,Aortic Stenosis, Pulmonary Stenosis
• Abdomen and pelvis- Urinary Tract anomalies, Absence of Ovaries
• Vagina and Uterus maldeveloped
• Normal Intelligence or Slight Retardation
• Cubits Valgus
• Defect in Arithmetic skill, Reading Maps, Drawing diagrams
• Sterile due to failure of development of ovary

Genotype – Monosomy of X chromosome (45,XO) ,Mosaicism-45,XO/46XX,

Isochormosome-46,X,i(Xq), Ring chromosome-46,X,r(X)

Sex chromatin or Barr bodies examination is Negative due to one X

chromosomes

Investigation required –

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 • Chromosomal analysis- to check karyotypen of patient , presence of Barr
body
• Plasma level of gonadotropins ,follicle stimulating hormone
• Radiological investigation to show renal and cvs abnormality

Counselling- Oestrogen replacement therapy should be given at adolescence

for development of secondary sexual characteristic. Affected female are sterile
but can bear child with the help of In Vitro Fertilization

Klinefelter’s Syndrome
It occurs in 1 in 1000 newborn individuals. Affected person is male with 47,XXY
genotype. First recognized by Harry Klinfelter in 1942

Incidence- 1 in 500 to 1,000 male births

Clinical Feature

• Patient attend clinic as married person for infertility

• Appearance of person is normal tall and with some degree of mental
retardation
• Barr body is present
• Genital system- Small testis but normal penis and scrotum, Azospermia ,
infertility
• CVS- pulmonary disease, breast cancer, varicose vein
• Sperm count are very less
• Puberty fails to occur normally
• Secondary sexual character doesn’t develop fully
• Scanty pubic and facial hair
• Gynacomestia

Genotype- karyotype 47,XXY, Mosaicism-46,XY/47,XXY with presence of Barr

body

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Counselling

• Hormone replacement therapy at puberty is advisable

• Testosterone will help in development of secondary sexual characters
• Married couple should be advised for various InVitro fertilization technique

Mutations-
Definition- A sudden heritable physicochemical change in DNA (gene) which
alters the effect on the character influenced by it

Mutation are classified in following ways

• On the basis of Gene Involved

• Point mutation- this type of mutation involve single gene
• Gross or Chromosomal mutation – this type of mutation involve one
chromosome or set of chromosome eg, polyploidy
• On the basis of Origin
• Natural- this mutation occur spontaneously due to natural cause
• Induced- this mutation occur as a result of man-made agents like
radiation, chemicals.
• On the basis of Change
• Deletion- a part of gene is lost in this type of mutation
• Insertion – it involve one or more chromosome and there is alteration in
sequence of gene
• Duplication- a piece of DNA abnormally copied
• Framshift mutation- any change in DNA leads to change in gene’s
reading frame due to change in code of amino acid
• Missense mutation- it involve change in one DNA base pair lead to chane
in amino acid for another protein

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• Nonsense mutation- it involve substitution of one amino acid for another
which lead to formation of different protein
• Rearrangement- in this mutation there is change in location of gene and
its sequence
• On the basis of cell type
• Somatic cell mutation- these mutations occurs in somatic cell of body.
Such mutation are not transmitted to offspring.
• Germ cell mutation- it occur in reproductive cell and transmitted to the
gametes and subsequent generation.
• On the basis of direction-
• Forward mutation- in this type change from original to abnormal
phenotype
• Reverse mutation- in this type change in to mutant to original phenotype
occurs
Mutagens – the factors responsible for mutations
• X rays
• Ultraviolet rays
• Temperature
• Chemicals
• Phenols ,Caffeine, Formaldehyde
Advantage and disadvantage of mutations
• Evolution of spices – good mutation bring evolution in specie
• To understand principle of inheritances
• Cell metabolism and their mechanism are understood
• Use In study of synthesis of antibiotics from microorganisms
• Life threatening
• Cause various abnormality or syndrome
• Majority of mutation aon does not have any therapy or it is
very costly

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Mendel’s Law
In 19th century Gorge Mendel’s experiments on plants have shown that the factors
which determine the characters do not Mix. His series of experiment gave law of
Inheritance of characters called as Mendel’s law

1. Law of Unit Characters- all Characters are unit and certain factors(Gene)
which regulate expression of these characters work together for expression
of particular character
2. Law of Dominance – Factor or Gene exist in pairs. The character that is
expressed in first generation is called Dominant and the character which
remain suppressed in first generation is called Recessive
3. Law of Segregation – this law state that each individual possesses two
factors (gene) for particular character. At gametogenesis each members of
one pair of gene separate from one another and goes to each gamete.
4. Law of Independent Assortment- This law state that members of different
gene pairs assort independently of one another at the time of gametogenesis.
Because of independent assortment new characters ,new combination are
produced in offspring

Autosomal Dominant Inheritance

• Gene responsible for inheritance are present on Autosomes
• Manifestation of disease even in single dose or Heterozygous condition

Characteristic of Autosomal Dominant Inheritance

➢ Males and Females are equally affected

➢ Affected gene is present on autosomes
➢ The disorder is seen in every generation without skipping
➢ 50% of offspring in each generation are affected
➢ An affected person will have affected parents, if appear first time then it is
due to mutation.
➢ New mutation always occur at time of gametogenesis
➢ Normal offspring do not transmit to the next generation

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 Autosomal dominant disorders
Huntington’s chorea, familial Hypercholesterolemia, Achndroplasia,
Neurofibromatosis, ABO blood groups, Polydactyly, Polycystic Kidney.

Autosomal Recessive Disorder

In this type of Inheritance affected gene should be in homozygous state (double
dose)

Characteristic of Autosomal Recessive Disorder

➢ Genetic disease occurs when affected gene is present in homozygous

condition
➢ A person with single mutant gen does not expres the disorder
➢ A Heterozygous individual with affected autosomal recessive trait is Carrier
➢ This trait is seen in same generation among Brothers andSisters siblings
➢ Its is not seen in previous generation and subsequent generation
➢ Male and females are equally affected
➢ Some common autosomal recessive disease eg- Phenylketonuria,
Alkaptonuria, Cystic Fibrosis, Schizophrenia, Spinal Muscular atrophy

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Sex linked Inheritance
X linked inheritance means sex linked inheritance . it could be either dominant or
recessive
X-linked Dominant Inheritance
• This disorder is due to mutant gene present on X chromosome
• Gene is dominant it express in heterozygous condition both sex
• Affected male transmit to all his daughters but none of the sons
• Eg – Vit D resistant Rickets, Hypophosphatemia, Xg blood groups

X- linked Recessive Inheritance

• Predominately males are affected
• Female have two x chromosome so an recessive traits only manifests when
genes are in double dose (Homozygous).
• X linked recessive traits are very rarely seen in females
• Heterozygous female will manifest disease but they are carrier
• Heterozygous female will transmit the gene to next generation
• Males have only one X chromosomes the mutant gene will manifest disorder
even in single dose called –Hemizygous for that allele.
• Disorder is transmitted through unaffected carrier females to their sons
• Affected male will transmit this gene to all his daughters who will be carrier
and will transmit to 50% of his grandsons.
• Eg – Duchene muscular dystrophy, Hemophilia,

Genetic Counselling
Genetic Counselling plays vital role in prevention of genetic disorders. In the
present era very few genetic disorders has treatment and it is very expensive. It is

19
better to prevent occurrence of genetic disease. A Genetic counsellor inform about
the present and future possibility of genetic disease.

Role of Genetic Counselling

• Planning Parenthood- Genetic counsellor predict characteristic of genetic

disease, future progeny and planning of family
• Genetic Inheritance- a counsellor asses probability of having child with
genetic hereditary disease and assist parents about the transmission of
chromosomal anomalies.
• Awareness- Counsellor should create awareness about merit and demerit of
late marriage. Genetic disorders like Down’s syndrome has a strong relation
ship with advanced maternal age
• Consanguineous marriage – in such case counsellor should discuss the
genetic transmission of recessive genes and its syndrome
• Radiation – counsellor should make people aware about hazards of
radiological investigation during pregnancy and unnecessary exposure of
gonads to radiation
• Prenatal Diagnosis – Counsellor should predict the occurrence of Genetic
disorder in high risk pregnancy and advice patient to go for prenatal
diagnosis of such disease
• Termination of Pregnancy- Counsellor can advise about the possible
medical termination of pregnancy in fetuses with hereditary genetic disorder
to prevent occurrence of genetic disease
• Drug Hazard-Counsellor should discusses possible drug hazard in patient
with genetic disease .eg Antimalarial drug can produce hemolysis in G-6 PD
deficiency
• Adoption – Counsellor has critical role in couple with genetic disease going
for adoption. Counsellor should discuss present and future mental, social and
legal complication of adoption.
• In Vitro Fertilization –Counsellor should discusses fertilization technique
IVF with couple having genetic disease

Quality of Good Genetic Counsellor

• Good Knowledge of principal of Genetics

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 • Awareness about the Genetic origin of disease
• Good Communication
• Tactful and Kind approach

Prospective Genetic Counselling - In this identification of heterozygous

individual by various screening method. This individuals are made aware about the
risk of having children with genetic disease when the genes are in Homozygous
condition This approach is very useful in known disease in various area and caste.
Eg Sickle cell anemia, thalassemia

Retrospective Genetic Counselling- Mostly Genetic Counselling are done

retrospectively. In this method sterilization and termination of pregnancy with
genetic disease is suggested

Recombinant DNA
Definition – it is an artificially synthesized DNA which is constructed by insertion
of foreign DNA in to DNA of appropriate organism so that foreign DNA is
replicated along with the host DNA.

It is formation of new DNA by combining DNA fragments from different sources.

In this procedure DNA molecule is broken at two segment to get specific required
DNA segment or gene. This segment is then inserted to another DNA molecule at a
desired position. DNA thus obtained is called Recombinant DNA and this process
is genetic engineering. Recombinant DNA is then allowed to multiply in host cell
to obtain multiple copies of specific DNA segment or gene

Recombinant DNA procedure

• DNA is cut into fragments by restriction endonuclease enzyme

• Incorporation of these fragments in suitable vector using DNA ligase
• Transformation of host organism by Recombinant Vector(plasmid) plasmid
is inserted into host cell by exposing the host bacterial cell into calcium salts
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 • Cell become permeable to plasmid
• Host cell containing vector and plasmid are put in a culture medium to
produce multiple copies (Clone)of foreign DNA fragments incorporated in
Recombinant vector
• Finally clones with proper DNA fragments are selected

Application of Recombinant DNA technology

• It gives approach to understanding of molecular basis of numerous disease

like Sickle cell anemia, familial hypercholesterolemia ,cystic fibrosis
• Using this technologies human proteins can be produced for therapeutic
purposes eg Insulin and Growth hormones
• Production of proteins for Vaccination eg Hepatitis B
• Proteins can be produced for diagnostic tests eg AIDS test
• Gene therapy for Sickle cell disease or Thalassemia
• Preparation of chromosomal maps and analysis of DNA sequence
• Production of monoclonal antibodies

Polymerase Chain Reaction

Cloning of DNA in vitro is done by Polymerase Chain Reaction called [Link]
machine can produce vast quantity of DNA fragments if we know bias pair
sequence of DNA. A small quantity of sample DNA can produce millions of
identical fragments in only couple of hours

Steps in PCR

1. Denaturation –double stranded DNA is converted in to single stranded by

heating DNA at 92-95˚C for 30 -60 seconds. Which separate two strand
without damaging DNA
2. Annealing- at temperature of 55-65˚C for 30-60 second s primer will anneal
with A=T and C=G base pairing to test DNA at specific location. High
concentration of primer prevent reannealing.
3. DNA synthesis – at about 72˚C new DNA strand is synthesized

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 All the 3 step occur in one cycle in PCR reaction. At end double quantity of
DNA is obtained .thermal cycle is programed to repeat PCR cycle 20 to 30
times in each cycle desired DNA is doubled so u get very fast results.

Advantage of PCR
• Small quantity of template is required
• Highly purified DNA sample is not required
• Number of sample can be used eg blood, bone, sperm, hair follicle
• It is used in Diagnosis ,Therapeutics, Criminology etc

Gene Therapy
Gene therapy is new way to treat disease. In this therapy defective gene is replaced
with normal gene to restore the lost gene function. With the help of gene therapy
abnormal gene is replaced with normal functioning gene. 1st gene therapy was
carried in 1990 by French Anderson on a child suffering from Adenosine
Deaminize deficiency (ADA) .Since then experiments are in progress world over
for 40 different types of gene therapy.

Two type of gene therapy

• Germ line gene therapy – In this gene therapy genetic changes could be
produced in both somatic as well as in germ cells. With this therapy changes
are transmitted to future generations. Due to moral and ethical issue this
therapy is unaccepted universally.

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 • Somatic cell gene therapy- In this gene therapy genetic changes are
targeted to particular tissue or organs. This is universally accepted.

Steps involved in Gene therapy

• Identification of defective gene

• Cloning of DNA sequence as well as controlling sequence which regulate
expression of that gene
• Cloned gene is introduce to vector
• Identification of target cell, tissue ,organ in vitro and transfer of genetically
corrected tissue to patient in vivo
• Insertion of normal functional gene in host DNA which involve transfer via
following methods
• Physical and chemical methods
✓ Microinjection of DNA in cell
✓ Electroporation – Cell membrane is made permeable with shocks
✓ Calcium phosphate precipitation
✓ Cationic lipid vesicle with DNA are introduced in cell
• Viral vector for gene transfer
✓ Most efficient mean of gene transfer
✓ Adenoviruses(DNA) and Retroviruses(RNA) are used
✓ Both method are stable and used in integrated therapeutic gene
therapy

Human Genome Project

Total DNA present in human cell is Human genome. In this project sequencing of
all DNA base pair is done and mapped on chromosomes. The HCP is publicly
funded international project of 15 year duration started in 1991 in USA. It is largest
project involved in 16 laboratories ,1,100 biologist, computer scientist, technicians
of 6 countries (UK, USA, France, Japan etc). Invention of new techniques the
Human genome project is completed

Objective of HCP

• Sequencing of human genome

• Preparation of Human genetic map
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• Mapping of Human inherited disease
• Development of New DNA technologies
• Development of Bioinformatics
• Functional genomics
• Comparative genomics

Finding of HCP
• Approximately 3.2 billion of bp in human genome with 26,000 to 35,000
gene present
• Gene constitute 5% of genome rest 95% is non-coding DNA( Junk)
• Non coding DNA junk are used as dating tools called “ repeats” based on
such DNA dating family trees of repeats prepared which give and
evolutionary history
• Bacterial gene are found in human genome
• One gene –one function theory gave new way to better genetic organization
theory
• 99% of DNA is same /alike in all human races. There is no superior race or
inferior race

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