Dr. G.K.

Chetan
Associate Professor, Department of Human Genetics, National Institute of Mental Health and Neuro Sciences, Bangalore – 560029.

MILESTONES IN GENETICS
YEAR
12,000 BC 8000 BC 4000 – 2000 BC 2000 BC 500 BC 16-18th AD Chinese Egyptians

SCIENTIST

CONTRIBUTION
Domestication of animals Domestication of crops like potatoes Used yeast for production of bread & cheese, fermentation of beer and wine Artificial fertilization of date palms 1st Antibiotics: moldy soybean curds used to treat boils Pre-Darwinian Theories of Inheritance - Blended inheritance - Acquired characteristics - Pangenesis

1859 1865 1869 1900

Charles Darwin & Alfred Wallace Gregor Mendel Friedrich Miescher Hugo DeVries, Carl Correns & von Tschermak Feulgan Frederick Grifith

Theory of Evolution “Experiments in Plant Hybrids” - established the basics rules of inheritance Identified Nuclein from pus cells Rediscovery of Mendel’s work

1927-28 1928

Demonstrated two types of Nucleic acids, i.e. DNA and RNA. Demonstrated genetic transformation principle.

1940 1944

Erwin Chargaff Oswald Avery, Colin McLeod & MacLean McCarty Watson and Crick Frederick Sanger David Baltimore, Renato Dulbecco & Howard M. Temin Sanger & Barrel, and Maxam & Walter Gilbert

Showed quantitative equivalence of total purine to total pyrimidines, i.e. A=T, G=C and A+T = G+C Demonstrated experimentally that DNA as the genetic material, they used Pneumococcus virulence and nonvirulent strains Structure of DNA Awarded Nobel Prize in chemistry for his work on primary structure of proteins; amino acid sequence of Insulin. Awarded Nobel Prize for their work on Reverse transcriptase and tumor causing viral activities Devised techniques for DNA sequencing.

1953 1958 1975

1975-75

OVERVIEW Function

Biochemistry

Genetics

Proteins Molecular Biology

Genes

HUMAN GENETICS

Incidence of Major Chromosomal Aberrations
ABERRATIONS Autosomes Numeric Trisomy 21 Trisomy 18 Trisomy 13 Other Trisomies Structural Balanced Rearrangements Unbalanced rearrangements Sex Chromosomes Female 45, X 47, XXX Others Male 47, XXY 47, XYY Others Total Chromosomal Aberrations 1:1000 1:1000 1:3000 1:160 1:10000 1 :1000 1:3000 1:500 1:2000 1 :800 1 :8000 1:20000 1:50000 Incidence : No. of Live Births

Incidence of Various Categories of Congenital Disorders
CHROMOSOMAL ABERRATIONS Overall Incidence Spontaneous Abortion 40-60% Late Still birth and Perinatal death 5-7% Live born

0.5%

Incidence of Main types of Chromosomal Abnormalities among Live Born Sex Chromosomal Abnormalities in males Sex Chromosomal Abnormalities in females Autosomal Disorders Balanced Translocations Unbalanced Translocations 1:400 1 :700 1:500-800 1 :500 1:2400

Genetic Diseases due to Single gene mutations
Overall Incidence among Live borns Autosomal Dominant, total Familial Hypercholesterolemia Huntington’s Chorea Deafness or Blindness Osteogenesis Imperfecta Autosomal Recessive, total Cystic fibrosis Phenylketonuria Mucopolysaccharidoses Glycogen storage disease X-linked disease, total Duchenne muscular dystrophy Hemophilia All other together 0.2-1.3% 0.6-7/1000 1:500 1:5000 1:10000 1:20000 0.9-2.5/1000 1:2500 1:12000 1:25000 1:50000 0.3-0.7/1000 1:7000 1 :10000 1:5000

Multifactorial Congenital Malformations
Overall Incidence of major malformations among live born 2-3% Spina bifida cystica Anencephaly Congenital heart disease Club foot Cleft Lip/Palate 1-4:1000 1-4:1000 6-8:1000 1-6:1000 0.15:1000

The Frequency of people with hearing Loss varies from 1.7% (in people <18yrs) to 34% (in people >18 and <65 yrs)

KARYOTYPING
• What is a Karyotype?
A Karyotype is an ordered arrangement of chromosomes. The Chromosomes are organized according to their size in descending order, position of centromere, and specific bands. In case of Humans, the karyotype is normally 46 XX in females and 46 XY in males

CHROMOSOMAL GROUPING
 Group A - Includes the largest Metacentric Chromosomes: 1,2 and 3  Group B - Includes the largest Submetacentric Chromosome: 4 & 5  Group C – Includes the large Submetacentric Chromosome: 6,7,8,9,10,11,12 and X (one or two depends on the sex)  Group D - Contains the largest Acrocentric Chromosomes: 13, 14, 15  Group E - Contains the smaller Submetacentric chromosomes: 16, 17 18  Group F - Contains the smallest of the Submetacentric Chromosomes: 19 and 20  Group G - Contains the small Acrocentric chromosomes 21, 22 and Y

(Human metaphase and karyotype)

Down’s Syndrome

(Trisomy 21 in person with Downs Syndrome)

Downs Syndrome with 14: 21 Reciprocal Translocation

This figure show a rarer form of DS (seen in 3-4%) . Here there is a reciprocal translocation between the 14th and 21st Chromosomes
t(14q21q)

Klinefelter's Syndrome

Karyotype of person with Klinefelter's Syndrome (47 XXY)

Turner’s Syndrome

Karyotype of Person with Turner’s Syndrome (45,XO)

Cri-du-chat Syndrome

Deletion of short arm of 5th chromosome in Cri-du-chat Syndrome (5p-)

Fragile-X Syndrome

Karyotype of person with Fragile-X (male)

TECHNIQUES

DNA Isolation
 Sources of DNA – DNA can be isolated from tissues like blood, skin, hair follicles, saliva and other bodily fluids. – It can also be extracted from Fossils and archival tissue like paraffin sections. – Mitochondrial DNA can also be isolated  DNA extraction – It is usually carried out using the phenolchloroform extraction method and the isopropanol method, the tube method and salting out of DNA is also done

Electrophoresis
This is essentially a separation method Molecules are separated based on the size (denaturing) and charge (native) as the travel through a porous medium i.e., the Gel. Generally DNA separation is done on Agarose gels and Proteins on Poly Acrylamide Gels (PAGE) In order to separate large DNA fragments (>500 kb) PFGE is used wherein the orientation of the field in continuously reversed to as to break the DNA into smaller fragments For refined protein separation 2D Gel Electrophoresis is done. Here the first Electrophoresis is done vertically and the second horizontally

Agarose Gel (DNA) PAGE

Pulse Field Gel Electrophoresis (PFGE) 2D Gel Electrophoresis

Different Types of Gel Electrophoresis

Polymerase Chain Reaction
A direct method for DNA Amplification There are three steps involved• • • Denaturing of the template DNA Strand (920 C) Annealing of Primers (50-550 C) Primer Extension (720 C)

The PCR Mix contains, dNTP, Primer, Buffer, Mg 2+, Taq Polymerase (or any other Thermostable Polymerase), Template DNA and Mineral Oil. There are different types of PCR - rtPCR (Reverse Transcriptase PCR), ARMS (Amplification Refractory Mutation System), Nested, Multiplex, DOP (Degenerate Oligonucleotide Primer) and qrtPCR (Quantitative Real Time)

Techniques of Molecular Biology
• Molecular Cytogenetics – FISH – SKY – CGH DNA Cloning – DNA Recombination Technology – Cloning Vectors Genomic Library and cDNA Library Gel Electrophoresis Blotting Methods and Applications Polymerase Chain Reaction (PCR) DNA Sequencing Production of Recombinant Proteins Organism Cloning Bioinformatics DNA Microarray Stem Cells based applications RNA interference

• • • • • • • • • • •

Fluorescent In-Situ Hybridization (FISH)
• The principle of FISH involves the hybridization of a labeled Oligonucleotide “Probe” to the target region on a particular chromosome, which is in a denatured state. This is used to identify chromosomal aberrations both Numerical and Structural. Also gene expansions and deletions can be identified

Types of FISH  Interphase FISH (nucISH),  Multicolor FISH (m-FISH),  Locus Specific Probes,  Satellite Probes,  Whole Chromosome Painting (WCP),  Multiband FISH (MCB),  Comparative Genomic Hybridization (CGH)  Spectral Karyotyping (SKY)

Fluorescent In-Situ Hybridization (FISH)
 This is a newer and more powerful technique for Cytogenetic analysis  The principle of FISH involves the hybridization of a labeled Oligonucleotide “Probe” to the target region on a particular chromosome, which is in a denatured state.  Specific chromosomal aberrations both numerical and structural can be identified.  Different types of analysis are available– Interphase FISH (nucISH), – Multicolor FISH (m-FISH), – Whole Chromosome Painting (WCP), – Multiband FISH (MCB) – Comparative Genomic Hybridization (CGH) and – Spectral Karyotyping (SKY)

nucISH showing normal number of Chromosome 13 (Green) and 21 (Red)

nucISH showing Trisomy 21 (three red signal) and two Chromosome 13

Metaphase FISH showing deletion in 15 Chromosome for the SNRPN Gene in a PWS positive case

Metaphase FISH for Alpha Satellite Region

Whole Chromosome Painting for Human Chromosomes

SKY of complex translocation involving chromosomes 8, 9, 22 in a CML Patient

CGH can be used to detect gene amplification and deletions This is done by hybridizing differentially labeled DNA to a sample This technique is widely used to study Cancer cases. In this case three differently labeled DNA samples were hybridized and their ratio is determined. Here one of the DNA will be of the affected type the other Normal. The ratio of the three shows which region is amplified and which is deleted.
CGH Showing Ratio metric Image of triple stained Chromosomes

DNA Sequencing
The term DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Different types employed: – Maxam - Gilbert sequencing: DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases. – Chain-termination methods:Use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators. 2007: Solexa and Applied Biosystems release next-generation sequencing technology with potential to produce 106-fold greater sequencing data than capillary electrophoresis systems.

Microarrays and Gene Chips
 This method is relatively new and is Based on CGH. It involves the hybridization of sample DNA onto a slide containing numerous genes or different forms of the same gene.  It simplifies the process of scanning for many gene in a single scan  Up regulation and down regulation of genes can be detected  Allelic forms of a gene can be detected

Microarrays: Principle and a typical Array

Nanotechnology
The main unifying theme is the control of matter on a scale smaller than 1 micrometre, normally approximately 1 to 100 nanometers, as well as the fabrication of devices of this size Highly multidisciplinary field, drawing from fields such as applied physics, materials science, colloidal science, supramolecular chemistry, biotechnology and even mechanical and electrical engineering

Nanotechnology applications
Clinical Diagnostics: lab-on-a-chip Tissue engineering Quantum computers: very fast computing, based on quantum physics Drug delivery – Nano porous materials could hold small drug molecules, transporting them to the desired location. – Applications include cancer treatment with iron nano particles or gold shells

Pharmacogenomics
• The study of how an individual's genetic inheritance affects the body's response to drugs. • Holds the promise that drugs might one day be tailormade for individuals and adapted to each person's own genetic makeup. • Environment, diet, age, lifestyle, and state of health all can influence a person's response to medicines • But understanding an individual's genetic makeup is thought to be the key to creating personalized drugs with greater efficacy and safety. • Pharmacogenomics combines traditional pharmaceutical sciences such as biochemistry with annotated knowledge of genes, proteins, and single nucleotide polymorphisms.

Anticipated benefits of Pharmacogenomics
• More Powerful Medicines • Better, Safer Drugs the First Time • More Accurate Methods of Determining Appropriate Drug Dosages • Advanced Screening for Disease • Better Vaccines • Improvements in the Drug Discovery and Approval Process • Decrease in the Overall Cost of Health Care

Barriers to Pharmacogenomics progress
• Complexity of finding gene variations that affect drug response • Limited drug alternatives • Disincentives for drug companies to make multiple pharmacogenomic products • Educating healthcare providers

ADVANCES

Applications in Health Biotechnology care – Pharmacogenomics
– Producing antibodies, Vaccines – Genetic manipulation to understand diseases Crop production and agriculture – Transgenic plants Non-food uses of crops – Bio fuels Environmental uses – Bioleaching – Bioremediation Industrial biotechnology: Breweries, Leather Food Technology Animal Biotechnology: Transgenics, Drug Development and Trials

Gene Libraries
 Libraries are repositories of DNA fragments cloned in their vectors  Libraries can be classified based on the cloning vector – e.g. cosmid, BAC, or YAC  Alternatively, the library can be described in terms of the source of the cloned DNA fragments. A. Genomic libraries: If total genomic DNA is digested and the fragments are cloned into an appropriate vector, this is a genomic library B. cDNA libraries: A cDNA (complementary DNA) library is generated from mRNA transcripts, using the enzyme reverse transcriptase, which can create a DNA complement to a mRNA template Since the cDNA library is based on mRNA, the library will represent only the genes that are expressed in the tissue and/or developmental stage that are sampled

Human Genome Project
Goals of the project were toIDENTIFY all the approximately 20,000 - 25,000 genes in human DNA, DETERMINE the sequences of the 3 billion chemical base pairs that make up human DNA, STORE this information in databases, IMPROVE tools for data analysis, TRANSFER related technologies to the private sector, and ADDRESS the ethical, legal, and social issues (ELSI) that may arise from the project

Stem Cells
Stem cells are primal cells found in all multi-cellular organisms They retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types The three broad categories of mammalian stem cells are:

– embryonic stem cells, derived from blastocysts – adult stem cells, which are found in adult tissues – cord blood stem cells, which are found in the umbilical cord

Properties of Stem Cells
# Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. # Pleuripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. # Multipotent stem cells can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). # Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells.

Stem cell therapeutics
Medical researchers believe that stem cell therapy has the potential to radically change the treatment of human disease A number of adult stem cell therapies exist, particularly bone marrow transplants that are used to treat leukemia In the future, medical researchers anticipate being able to use technologies derived from stem cell research to treat a wider variety of diseases including cancer, Parkinson's disease, spinal cord injuries, and muscle damage, amongst a number of other impairments and conditions Stem cells, however, are already used extensively in research, and some scientists do not see cell therapy as the first goal of the research, but see the investigation of stem cells as a goal worthy in itself There still exists a great deal of social and scientific uncertainty surrounding stem cell research, which could possibly be overcome through public debate and future research

Gene Therapy
 Gene therapy is an experimental technique that uses genes to treat or prevent disease  In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery  Researchers are testing several approaches to gene therapy, including: – Replacing a mutated gene that causes disease with a healthy copy of the gene. – Inactivating, or “knocking out,” a mutated gene that is functioning improperly. – Introducing a new gene into the body to help fight a disease.

Types of Gene therapy
Gene augmentation therapy This is appropriate for the treatment of inherited disorders caused by the loss of a functional gene product which is solved by adding a functional copy of the lost gene back into the genome Gene inhibition therapy This is suitable for the treatment of infectious diseases, cancer and inherited disorders caused by inappropriate gene activity. The aim is to introduce a gene whose product inhibits the expression of the pathogenic gene or interferes with the activity of its product. Killing of specific cells This is suitable for diseases such as cancer that can be cured by eliminating certain populations of cells. One approach is the expression of an enzyme that converts a harmless prodrug into a highly toxic molecule. Another is the expression of a protein that makes the cells vulnerable to attack by the immune system.

RNA Interference
 This is a naturally occurring defense mechanism against dsRNA. It was also found to play regulatory properties in various cellular processes.  As it acts on the mRNA level it is also known as Post Transcriptional Gene Silencing (PTGS).  The mechanism of RNAi was demonstrated in C. elegans by Fire et al, 1998  Another technique that is similar to RNAi is the use of “Antisense Oligonucleotides”

Components of RNAi Pathway
The two main types of RNA that are involved in this pathway are short interfering RNA (siRNA and rasiRNA) and microRNA (miRNA) Synthesis of siRNA involves processing of dsRNA into short 21-22nt long fragments with characteristic 3’ 2nt overhangs. This is done by the Dicer enzyme (A type 3 RNase). These siRNA are then integrated into the RNA Induced Silencing Complex (RISC) which then degrades target mRNA. Another set of enzymes that can endogenously produce dsRNA as well as use siRNA as primers for dsRNA production are RNA dependant RNA Polymerases (RdRP)

FUTURE

Other frontiers of biotechnology
ELSI (Ethical, Legal and Social Issues) Patents and IPR (Intellectual Property Rights) Cheminformatics NDDS (Novel Drug Delivery Systems) Stem Cell Repositories Phylogenetics: evolutionary distances Transgenics Clinical Trials

After B. Sc

Combined Biotechnology Entrance Exam for admission into MSc. Biotechnology, M.Sc (Agri) and M Tech in Biotechnology is conducted by Jawaharlal Nehru University New Delhi for 29 different participating Universities Joint Admission Test to M.Sc. – JAM is conducted by the Indian Institute of Technologies – IITs for admission to Master of Science ( M.Sc.) and other post–B.Sc. programmes offered at the 7 different Indian Institutes of Technology (IITs) IISC Integrated Ph D program in 4 disciplines (Biological, Chemical, Physical & Mathematical Sciences) NCBS Integrated Ph D program is for graduates with a Bachelor's degree in any branch of basic science NCBS MSc. This programme is for graduates with a Bachelor's degree in any branch of basic sciences. They can opt either for M.Sc programme of DBS or for both (Integrated PhD programme of NCBS/DBS or DBS programme of M.Sc.)

Career prospects

After M. Sc.

Career prospects

IISC Ph D program in 27 departments (www.iisc.ernet.in) MPhil: A one/two year course. This has a set curriculum which includes a dissertation project. Such courses are offered by various Universities such as JNU, BHU (Varanasi), NIMHANS. MBA in Biotechnology: 2 year course in management training pertaining to the field of Biotechnology. Offered by The University of Pune MBA in Pharmaceutical Marketing: 2 Years course offered by NIPER (National Institute of Pharmaceutical Education and Research ) Chandigarh. Advanced Diploma in Bioinformatics offered by JNU, MKU, Jadavpur Univ. (WB), IIIT (Allahabad) and BHU (Common Entrance Test) Ph.D: Students with a Master's degree in any branch of science (e.g. Physics, chemistry, mathematics, biology) in any applied science (e.g. engineering or medicine) are eligible. Scholarships are offered by CSIR- UGC NET, ICMR, ASRB (Agricultural Scientist Recruitment Board), DRDO etc.

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