Recombinant DNA technology uses restriction enzymes and other tools to combine DNA fragments from different sources and insert them into vectors like plasmids. This allows genes to be cloned and mass produced. Key applications include producing human insulin to treat diabetes, vaccines like for hepatitis B, and gene therapy. Plasmids are commonly used vectors that are small, self-replicating DNA molecules found in bacteria. They contain origins of replication, antibiotic resistance genes as selectable markers, and sites for inserting foreign DNA. Recombinant DNA technology has proven important for developing medical treatments and furthering pharmaceutical research.
DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.
introduction
What is virus
What is virus resistance plant
History
Gene use for develop virus resistance plant
Coat protein gene
cDNA of satellite RNA
Defective viral genome
Antisense RNA approach and
Ribozyme – mediated protection
conclusion
References
This is about methods of creating transgenic animals,applications of transgenic animals in biotechnology and application of transgenic animals in pharmaceuticals.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.
introduction
What is virus
What is virus resistance plant
History
Gene use for develop virus resistance plant
Coat protein gene
cDNA of satellite RNA
Defective viral genome
Antisense RNA approach and
Ribozyme – mediated protection
conclusion
References
This is about methods of creating transgenic animals,applications of transgenic animals in biotechnology and application of transgenic animals in pharmaceuticals.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
Transgenic manipulation of animal embryos and its applicationDeveshMachhi
INTRODUCTION
Genetic manipulation in animal for higher productivity is also called genetic engineering, refer to the alteration of the gene of an organism.
Organisms containing integrated sequences of cloned dna (transgenes), transferred using techniques of genetic engineering (to include those of gene transfer and gene substitution) are called transgenic animals.
Transgenic technology has led to the development of fishes, live stock and other animals with altered genetic profiles which are useful to mankind.Genetically modified animals are proving ever more vital in the development of new treatments and cures for many serious diseases.
Transgenesis is a radically new technology for altering the characteristics of animals by introducing the foreign genetic material.
CONTACT: devmac1323@gmail.com
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
this helps to understand the normal techniques related to biotechnology in a simple manner and provides you broad idea about the subject. A brief knowledge about the topic is presented in this presentation.
This Power point presentation describes various cloning strategies especially isolation of desired DNA/Gene to be cloned. It describes isolation of DNA/gene to be inserted in vector in 5 different situations.
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
Transgenic manipulation of animal embryos and its applicationDeveshMachhi
INTRODUCTION
Genetic manipulation in animal for higher productivity is also called genetic engineering, refer to the alteration of the gene of an organism.
Organisms containing integrated sequences of cloned dna (transgenes), transferred using techniques of genetic engineering (to include those of gene transfer and gene substitution) are called transgenic animals.
Transgenic technology has led to the development of fishes, live stock and other animals with altered genetic profiles which are useful to mankind.Genetically modified animals are proving ever more vital in the development of new treatments and cures for many serious diseases.
Transgenesis is a radically new technology for altering the characteristics of animals by introducing the foreign genetic material.
CONTACT: devmac1323@gmail.com
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
a brief description on the new emerging genome editing technology CRISPR-Cas9. this technique is making its place stronger and stronger day by day. and impossible things can be possible by this technique. and some main and famous names who discovered this technique.
this helps to understand the normal techniques related to biotechnology in a simple manner and provides you broad idea about the subject. A brief knowledge about the topic is presented in this presentation.
This Power point presentation describes various cloning strategies especially isolation of desired DNA/Gene to be cloned. It describes isolation of DNA/gene to be inserted in vector in 5 different situations.
A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms.
More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host.
Recombinant DNA molecules are produced with one of the following objectives:
1. To obtain large number of copies of specific DNA fragments.
2. Large scale production of the protein encoded by the gene.
3. Integration of the desired DNA fragment into target organism where it expresses itself.
Drought tolerant-genetically modified plants:
Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants
Of all abiotic stress, drought is regarded as the most damaging
Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management
Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success.
Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions.
Unlike conventional plant breeding there is no need of repeated back crossing
Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved.
Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
Assignment on Recombinant DNA Technology and Gene TherapyDeepak Kumar
Assignment on Recombinant DNA Technology and Gene Therapy Basic principles of recombinant DNA technology-Restriction enzymes, various types of vectors, Applications of recombinant DNA technology. Gene therapy- Various types of gene transfer techniques, clinical applications and recent advances in gene therapy
This is one of the major chapters for the examination NEET. A few questions are expected from this chapter and carry more weight as per the NEET syllabus.
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R dna technology, biotechnology
1. BIOTECHNOLOGY
CLASS-XII
Recombinant DNA Technology-
Science uses various techniques to treat disease and improved their environment.
There are various techniques, out of which some techniques are used known as
Gene Cloning and RDT.
Previously this technique is used in production of human insulin (Diabetes).
For eg. Yeast cells- Hepatitis B vaccine
Plants such as Bt- Cotton (Insect resistant)
First time rDNA molecules generated by-Paul Berg, Herbert Boyer, Annie Chang
and Stanley Cohen in 1973.
These are important to produce large amount of rDNA (Gene cloning)
Large amount of protein expressed by insert.
These are important in today’s medical practice, essential to Pharma industry,
help to diagnose genetic disease such as Sickle cell anemia or used in
development of vaccine.
Recombinant DNA technology proved its importance in the production of vaccines
and protein therapies such as- Human insulin, interferon and human growth
hormone.
These are also used to produce clotting factor for treating Haemophilia and in the
development of gene therapy.
Application- In nutrition, in medicine to create pharmaceutical products (Human
Insulin).
# Importance of Plasmid-
These are relatively small DNA sequences that can self replicate and exist
independent of the chromosome.
2. Also carry antibiotic resistance genes that make them selectable.
Used to produce large number of copies at low cost.
Examples- Escherichia coli cells can be stored with the genes for human insulin in
their chromosomes.
Pharmaceutical products.
Vaccines
Diagnostic testing
Gene therapy
DNA finger printing
DNA and Agriculture
Types of Plasmid- there are various types of plasmids such as,
Fertility plasmid
Resistance plasmid
Virulence plasmid
Degradative plasmid Types
Col plasmid
Tools of rDNA Technology-
Restriction Enzymes :
The molecular scissors
Foundation of rDNA technology
Enzyme exist in many bacteria function as defense mechanism called restriction
modification system.
There are three types of restriction enzymes-
Type I, II, III
Out of which only Type II restriction enzyme is used in rDNA technology.
It recognize and cut DNA with in a specific sequence typically consisting of 4-8 bp.
Sequence referred to restriction site is generally Palindromic, both strand same.
Type II named on the basis of isolation of bacteria,
Eco RI
where E represents Genus,
co represents species,
3. R represents strain (RY 13)
I represents isolated for the first time
First discovered by – W, Arber, H.Smith and D.Nathans in 1978 and awarded by
Nobel prize
Table: types, microbial sources, recognition sequence- Ref. NCERT XII
Figure: Construction of rDNA using fragments from different sources,
Ref. NCERT XII.
Blunt end and Sticky end
DNA restriction sites cleaved by restriction enzymes results in
complementary ssDNA called Sticky ends.
Production of no complementary ssDNA results in blunt ends.
Restriction fragment length polymorphism (RFLP)-
DNA of each organism has specific sequence which can be cleaved by
several restriction enzymes and fragments of different length can be
produced.
These fragments are called restriction fragments.
Restriction fragments of different individuals and species are different.
These are of different length because of variation in DNA sequence of
restriction sites.
These variation are called RFLP.
Diagram
(Ref. NCERT XII)
Application of RFLP-
It is mostly used in DNA finger printing analysis technique.
Other enzymes used in rDNA Technology-
There are various enzymes that are used in rDNA technology
4. DNA Pol I -
a) It synthesizes DNA complementary to DNA template in 5’-3’
direction.
b) It lacks 5’-3’ exocatalytic activity.
Exonuclease III –
It cleaves from the end of a linear DNA and digest dsDNA from the 3’ end
only.
DNA ligases- Role in sealing ss nicks in ds DNA.
Forms phosphodiester bond between two adjacent nucleotide.
There are two types-
a) E.coli DNA ligase- Uses NAD as a source of energy.
b) T4 DNA ligase- Uses ATP.
Alkaline Phosphate (AP)-
a) 5’ phosphate group is essentially required for ligation of DNA fragments.
b) DNA can’t be ligated if 5’- phosphate group is removed.
c) The enzyme removes 5’ Phosphate group from 5’ DNA fragments and make
it free.
d) Enzyme is used for self-ligation.
There are different sources, Bacteria (BAP) and Calf Intestine (CAP).
Reverse Transcriptase-
RNA dependent DNA polymerase synthesizes DNA complementary to a RNA
template in 5’-3’ direction.
RNase A-
It is a nuclease which digest RNA but not DNA.
Tag DNA Polymerase-
DNA Pol isolated from a thermophilic bacteria (Thermus aquaticus)
Operates at 72 degee celcius.
Stable above at 90 degree celcius.
Used in PCR.
Terminal Transferase-
Enzyme adds several nucleotides to 3’ end of a linear ds DNA or RNA.
5. SI Nuclease-
SI nuclease acts on single strands of double stranded DNA and result blunt ends in
DNA fragments.
Vectors-
These are the vehicle used for cloning.
For eg. Plasmid
Features of vectors-
Ori (independently replicate with in host).
Incorporate a selectable marker.
Identify host cell containing vector.
Selectable Marker-
It include gene conferring antibiotic resistance.
Enzyme such as beta-galactosidase.
Gene expressing green fluorescent protein (GFP).
Easy to viewed under UV light.
Vector must have one unique restriction enzyme recognition site, used for
cutting and introducing an insert.
More than onr restriction site.
Contain a multiple cloning site (MCS) or Polylinker.
Provides flexibility in the choice.
It should be small in size so that entry into host cells.
Features-
It should easily be isolated from the organisms.
It should be small,
Larger vector DNA get broken during purification.
Origin of replication.
Contain a multiple cloning site.
Selectable marker (Helps to select the host cells from population).
Examples of Common selectable markers-
Genes providing antibiotic resistance
6. Eg. Ampicillin (ampr
), tetracyclin (tetr
), kanamycin (Kanr
).
Beta-galactosidase (Presence of this enzyme is identified by the change in color of
substrate).
Cloning vector Genome size (kb) Insert size (kb)
Bacteriophage lamda 48.514 9-23
Cosmids 30-45
pBAC 300-500
pYAC 1,000-2,500
Plasmid 1-1.50 0.5-8
M13 6.407 Less than 5
Plasmids-
Plasmids are extra-chromosomal
Self replicating
Circular
ds DNA molecules
Found in bacteria and yeast
Plasmids are not essential for normal cell growth & division.
Earliest plasmid vector
pBR322
contain two different antibiotic resistance genes recognition sites for several restriction enzymes
Shuttle vector-
Plasmid vectors can replicate only in Escherichia coli.
Many of them are used in eukaryotic cells.
They can exist in both.
In eukaryotic and in Escherichia coli such vectors are known as shuttle vectors.
Example of shuttle vector-
Yeast Plasmid Yep,
Naturally occurring plasmid of the bacterium ,
Agrobacterium tumefaciens called Ti plasmid.
7. Vectors based on bacteriophages-
Bacteriophages are viruses that infect bacterial cells by infecting their DNA
into them.
Take over the machinery of bacterial cells to multiply themselves.
DNA is selectively replicated and expressed in the host bacterial cell.
Resulting in number of phages out of the cell by lytic pathway .
Infect neighbouring cells.
Ability to transfer DNA from the phage genome to specific bacterial host during
the process of viral infection.
Two phages that have been extensively modified for the development of cloning
vectors are-
a. Lamda
b. M13 phages