250
SaraSEUblicaionPublisher
Responders
Pharmacogenomics
Pharmacogenomics
Patients with different genotypeS Rapid Responders
Non Responders
Fig. 94.1: Principle of pharmacogenomics.
Advantages of Pharmacogenomics
"Pharnacogenomics helps to identify the patients most likely to respond to
aparticular drug.
"It provides hope to physicians for better medication selection for Datiente
"For disorders like cardiovascular disorder, different drugs exist in the mar-.
ket, each having diferent effects on different patients. Hence researches willbe
able to categorize such drugs for the right patient.
Human
95 Genome Project
The human genome project is a multinational research project to
determine the genomic structure of man (Homo sapiens). It is aiming at
sequencing allDNAs of man and at determining the location of variousgenes in
the DNAs. Many Government and Private sectors took part in the project.
The genome project was initiated in 1988 and got completed in 2003. It
was under the International Administration of the Human Genome Organiza
tion' (HUGO). It was funded by the Department of Energy(DOE) and Na
tional Institute of Health (NIH) in the USA, the European Commission
(EC)and Britain's Welcome Trust.
The research works in the project was conducted in research laboratories
in six nations. The most important among them were the National Human
Genome Research Institute (USA) and Sanger Centre (England).
GENETICS 251
Methodology
Asomatic cell ofhuman being contains 23 pairs of chromosomes. Each
chromosome;is composed of along
double-stranded!i DNA and histone protein.
estimatedthat there are 3.2 million
jis basepairssiin the DNA ofall these chromo-
[Link], 2 million basepairs are already sequenced. The general
thodology of thegenome project is outlined below:
Obtaining DNA
A
cell culture containing ahuman celline is homogenized in sucrosesolu
tion usinga pestle.
The homogenate so obtained is subjected to differential centrifugation
achromosomal fraction.
to getThis fraction is added with a lysis buffer and again centrifuged using an
sizes. Thus
ultracentrifuge to separate different chromosomes according to their
human chromosomes 1to 23are isolated and numbered properly.
histone proteins.
In the chromosomes, DNA is wrapped around ball-like
nucleosomes.
These DNA wrapped units are called containing Tris-HCI, EDTA
Each chromosome, is suspended in a solution releases histones from the
treatment
and NaCland is stored at-20°C. This
nucleosomes, releasing the DNA free. chloride density gradient cen
The DNA is isolated from it by Cesium
long.
trifugation. Each DNA is about 5 feet
Late prophase nucleus
Homogenation and |
Centrifugation
Nuclei
Lysis bufterand
Human cell
ultracentrifugation
Chromosomes
U USeparation solution
DNA and ultracentrifuga
tion
DNA
Histone
study
Obtaining DNA forgenome
Fig.95.1.
252
SaraSublication
Biosiences Book Publ
Preparation of DNA for Study
gDNAofa chromosomeiscutintosmall pieces of 5000 -10,000
Thelong enzymethat cutsthe DNA at long distances,
nucleotides
The individual restriction
using apieces ofDNAare separated by agarose gel electrophoresis
on the basis of restriction:nfragmentlengthpolymorphism(RFLP) E
Each andevery
stored at 20°Cfor-
vialand
DNAfragment so obtained, is placedin a plasmid DNAto futurea use.
One DNA is inserted
fragmentiinto into a construct rDNA
The rDNA is introduced [Link] invivoamplification of that)DNA frag-
DNA fragments are made, In th
ment. Consequently bilions of copies of that
way cach and every DNAfiragment is amplified.
Sequencing and Analysis
amplified rDNA is isolated from the bacterial cells and the target DNAi
The SmallDNAfragment
separated. Itis cut with arestriction enzyme to generate
R site
R sitea
DNA
Restriction
digestion and
Electrophore
SIS
Plasmid
DNA joining
and ligation
Freeze storage of DNA DNA
fragments at-20°C fragment
rDNA
[Link]
rDNA
Amplification ofDNA fiagment by
gene cloning in [Link]
Fig.95.2. Preparation of DNAfor genomic study.
GENETICS
253
tye
visible under laser light is added to thetterminalnucleotide
Fluorescent
DNAfragment. The resulting DNAsolution is pouredlinto
each
i 96tubes inside
sequencing machine. In the tubes, the DNA fragments are electro-
DNAveryfast and this can be observed by afluorescencei recorderinthe
phoresed
gne
mMachne
a
overlapping segments are identifiedland assembledin
Thebases in the
order,by using computer database. In this way, all DNAfragments of a
incar
chromosome,are sequencedto recreate its original nucleotide sequence.
study is conducted on all 23 chromosomes of human genometo
Such a
the exact genome structure of man.
understand
Applications of Genome Project
project provides database information of DNA sequences of
LGenome information to manufacture
based companies may use the
oon Biotechnology disease treatments.
proteins which are of much use in the human inheritance. 289
human disorders in man and their
[Link] helps to detect genetic Examples:
known.
cenetic diseases have been Gaucher disease
GBA gene
a) ChromOsome I: Prostrate cancer
HPC 1gene - Alzheimer's disease
PS 2(AD4)
Loss of memory
Chromosome 2: CREB
b) Waardenburg syndrome
PAx3 (Mismatchingincolours)
Spinocerebellaratrophy
c) Chromosome 6: SCA 1gene (Loss ofcontractión)
kidney
Diabetesassociated with
IDDM l gene
failure
EPM 2A Epilepsy
Diabetes
d) Chromosome 7: GCK gene Cysticfibrosis
CFTR
Obesity
OBgene Refsumdisease
10:PAHX gene Gyrateatrophy
e)Chromosome OATgene (Progressivelossofvision)
Breastcancer
SevereCombinedimmunodeficiency
17:BRCA l
1}Chromosome Testisdifferentiation factor
20:ADA 1
8) Chromosome
Chromosome Y: SRY(TDF)
9)
254
Restriction
digestion and
Electrophoresis
Caraslication
Cut with
restriction
enzyne
abllsher
Numerous
copies of DNA,
fragment
Fluorescent
dye treatment
Pouring into
96 tubes in a
gene machine
A
Electrophoresis
Fluorescence recorder
Computer
..ATGACTCAGTA..
" , A Display Nucleotide sequence
Fig. 95.3. DNA sequencing and analysis.
[Link] remedial gene can be chosen and administered to treat genetic
disease.
[Link] action of harmfulgenes is blocked by introducing an antisense gene
to stop the genetic disease.
[Link] American company, Incyte Genomic has manufactured agene chip
with 10,000gene kits. This chip can be used to detect geneticdiseases, infec
tious diseases, oncogenes, parasitic worms, etc. at once during clinical diag
nosis. The diagnosis is very fast; it will be done within 3hrs.
6. By matching, the human genome with the genome of [Link]
tists conclude that this fly has remedialgenes for 177 genetic diseases in man.
7. The information about the genome willbe utilized todesign babies wti
many superior characters such as skill, strength and free of genetic disorders.
