Automated DNA
sequencing
Mechanism
All mechanism is same except
No need of radio labeling
Fluorescent labeled ddNTPs make the work easy
UV laser detectors are used
ddNTPs are labeled with special dye
When laser strike the colour a specific spectrum is excited
They are detected on photoelectric device
Data is fed in computer
Computer give the sequence of nucleotides on DNA
Maxam-Gilbert Method
1976 -1977 Allan Maxam and Walter Gilbert
Method:
Chemical cleavage method
Procedure :
DNA 5`end labeled through kinase treatment with P32
Kinase transfer radioactive P from ATP to 5` end of DNA
Dimehtylsulphoxide is added and labeled DNA is heated to 90
C
DNA denatured to two single strands
Separation of strands with gel electrophoresis
Formation of four samples i.e. G,A + G , T+ C and C
Then the DNA is treated with specific cleavage reagents and
DNA analysis
Emerged in 1980by Alec Jeffery
Made first DNA finger printing in 1985
Help in the recognition of organisms
Procedure:
Treatment with restriction enzymes
Different length DNAs are obtained
Separated with gel electrophoresis
And read from the gel
Collection of DNA samples : Steps
Collected from blood or hair root
Amplified through PCR
Placement of RFLP:
Different sized DNA fragments created through the same restriction
enzyme
RFLPs of any two individuals are different from one another
99% DNA is same while 1% is different from one another
Separation of RFLPs:
Through gel electrophoresis
Southern blotting :
Transfer of fragments to a filter paper
Detection by probe hybridization
Autoradiography:
Extra probes are washed
Pattern of hybridization is observed on X-ray film
Application of DNA analysis
Criminology
Catastrophe victims identification
Family relationship
Identification of endangered species
Organ donors matching
Pedigree
Genome maps
To find the number of genes in an individual
Loci of particular on chromosomes
Number of genes on each chromosome
Age at which particular genes become on
Genetic markers
DNA sequence with a known location on a chromosome
that can be used to identify individuals or species.
Visible marker:
Can easily be observed as eye colour
Invisible marker:
Non coding region of chromosome used to detect a gene
Common markers:
RFLP , VNTRs , Microsatellite polymorphism . SNPs
Human genome project
Started in 1900 and completed in 2003
Director of institute:
James D Watson
Funded :
US government and later Welcome trust
Major goals:
Identify all genes i.e. 20,000 to 25,000 genes
Sequencing of 3 billion nucleotides
Store information in databases
Data analysis
Transfer related technology to private sector
Address the ethical , legal and social issues
Benefits of HGP
Benefits :
Diagnosis of disease
Earlier detection of disease
Gene therapy
Pharmacogneomics
Rational drug design
Tissue culture
Development of a plant from a cell or tissue
Explant:
Plant part from which cell is taken
Steps :
Taking cell from the explant
Sterilization of explant
Media preparation
Inoculation
Addition of more Cytokinin and low auxin
Development of callus
Application of auxin for tissue differentiation
Formation of a plant
Animal cell culture
Culturing animal cell on artificial media
Uses:
Recombinant DNA technology, genetic manipulation , industrial
processes
Vaccines , monoclonal antibodies , pharmaceutical drugs and cancer
research
Techniques:
Primary cell culture:
Cell removed from animals body is cultured
Secondary cell culture:
Cell removed from primary culture and cultured
Histotypic culture:
Culturing for tissue formation
Organotypic culture:
Requirements
Incubator
Synthetic media with vitamins , amino acids , and fetal calf
serum
Application of animal cell culture:
Antiviral vaccines
Genetic manipulation
Monoclonal antibodies
Pharmaceutical drugs
Chromosomes analysis
Study of effects of toxins
Use of artificial skin
Study of function of nerve cells
Transgenic lamb was developed in 2000
Transgenic organisms
Having foreign gene also called as GMOs
First transgenic organisms:
Bacteria ( [Link])
Transgenic bacteria:
In 1978 by Herbert Boyer
[Link] for the production of insulin
Role of transgenic bacteria :
Insulin for diabetic patients
Clotting factors for treating haemophilia
GH for treating dwarfism
Tissue plasminogene activator to treat thrombotic
disorder
Interferons to treat viral infections
Old products were purified from cadavers and was the
Ecological concerns with transgenic bacteria
Production of new pathogen
Transgenic plants
First transgenic plant:
Tobacco plant
The plant is resistant to many pests
France and USA in 1986
Methods:
Gene of interest is inserted in a single cell and cultured through
tissue culture
First gene for pests resistant :
Cry gene from [Link] also known as Bt gene
Benefits:
Tolerant against biotic stress i.e. bacteria , viruses , pests , weeds
etc and abiotic stress
Delay fruit ripening
Examples:
Roundup ready , soybean , freedom ii squash , high lauric , canola .
Registration: 1955
Transgenic animals
Animals with foreign gene inserted in its
genome
Benefits :
Production of more wool
More milk
Production of spider silk
Methods:
DNA microinjection
Embryonic stem cell mediated gene transfer
Retrovirus gene transfer
Biotechnology and human
welfare
Through biotechnology we can learn more about human
health so new techniques are developed to improve
human life
Development of vaccine:
Three different ways
1. Separation of pure antigen using specific monoclonal
antibodies
2. Synthesis of antigen with cloned gene
3. Synthesis of peptides to be used as vaccines
Through the knowledge of biotechnology we can recognize
diseases
Monoclonal antibodies
Same type of antibodies produced by the clones of one
parent cell
Production:
Mixing myeloma cells and spleen cells
This technique is somatic cell hybridization
DNA/RNA probes
Florescent labeled single stranded DNA or RNA
The sequence is already known
Use:
Genes are detected from genomic libraries
Diagnosis of diseases
Monoclonal antibodies are used in serological tests
Examples:
Herpes
Ready made DNA probes are also used for diagnosis
of disease
Cystic fibrosis
Inherited disease
Mucous membrane is affected
Problems in lungs and digestive systems
Defect:
Cystic fibrosis transmembrane conductance
regulator
Gene that regulate protein for the movement of
salts and water is defective
Gene therapy for cystic fibrosis:
1989 as it was the only possible solution of
cystic fibrosis
