18-12-2010, 10:40 AM
[attachment=7398]
Presented By:Mayank Bhardwaj
Historical Background
Father of Genetics – Gregor John Mendel
- Formulated set of rules to explain inheritance of biological characteristics.
- Basic assumption: Each heritable property is controlled by a factor, Gene.
- Rediscovery of Mendel’s law in 1900 marked birth of Genetics.
- Gene reside on chromosome: W. Sutton (1903)
- Experimental Backing: T.H. Morgan(1910)
- DNA as genetic material: Avery, McCarty, McLeod(1944), Hershey, Chase(1952).
- Between 1952-1966: Structure of DNA proposed, Genetic code cracked, Process of transcription and translation described.
Until decade of 1960 experimental techniques were not sophisticated enough to study gene.
- 1971 to 1973: Revolution in experimental biology.
# Recombinant DNA technology
# Genetic Engineering
Genetic Engineering A Biotechnological Revolution
The synonyms-
Recombinant DNA technology
R-DNA technology
Gene cloning
Molecular cloning
The Applications
Development of GMO and GEO.
Gene therapy (e.g. HGH, Human insulin).
Improving nutritional value of food.
Improving storage life of food.
Controlling water and air pollution.
DNA fingerprinting.
etc..
Gene cloning General process
Fragment of DNA with desired gene inserted to circular DNA molecule (vector) to produce chimaera / r-DNA
Vector act as vehicle for transferring gene to host cell ( gen. bacterium)
Within host cell, vector multiplies producing identical copies. Host cell itself divides and passes r-DNA to progeny.
Copies of desired genes are further extracted by selection and screening process.
Gene cloning Main Steps
Isolation and purification of DNA segments.
Addition of desired gene to vector.
Insertion of recombinant plasmid to host cell.
Selection and screening of clones of r-DNA
Gene cloning Basic Requirements
DNA purification from living cells for desired genes and vectors.
Vectors to carry the desired gene.
DNA manipulating enzymes.
Recombinant Gene transferring modes.
Recombinant Gene selection and screening modes.
Gene cloning 1. DNA purification
Two distinct kind of DNA are purified
a). Total cell DNA: i.e. desired DNA,
donor may be bacteria, animal, plant or any other cell.
Methods used are
- Centrifugation
- Phenol extraction
- Ethanol precipitation
- CTAB method for DNA from plant cell.
Gene cloning 1. DNA purification
B). Vector DNA : vehicle of desired DNA,
Cultures are prepared from plasmid or phages depending upon requirement.
methods used are
- Anion exchange column chromatography
- Alkaline denaturation method
- CsCl density gradient centrifugation
- PEG precipitation ( for phages).
Gene cloning 2. Vectors
It is a DNA molecule carrying foreign DNA to host cell, where it replicates producing its identical copies with foreign DNA.
- Also called Vehicle.
Types of Vector:
Plasmid
Phage/ Phasmid
Cosmid
Yeast artificial chromosome
Bacterial artificial chromosome
Gene cloning 2 a) Vector-Plasmid
Extra-chromosomal, circular DNA in bacteria, having self replicating abilities outside the host cell.
Cloning limit- 0.1 to 10KB.
Gene cloning 2 a) Vector-Plasmid-Types
Classification based on main characteristics coded by plasmid genes.
Fertility (F) plasmid: carry tra genes, have ability for conjugal transfer of plasmid. e.g. F of E.Coli
Resistance ® plasmid: carry gene for antibiotic resistance for host bacterium. e.g. RP4 of Pseudomonas.
Col Plasmid: Colicin code-protein killing other bacteria. E.g. ColE1 of E.Coli.
Degradatice plasmid : allow host bacterium to metabolize unusual molecule as toulene, salicylic acid.
e.g. TOL of Pseudomonas putida.
Gene cloning 2 b) Vector – Phage/ Phasmid
Virus infecting bacteria.
Affect bacteria by lytic and lysogenic cycles.
Although many varieties are there, mainly used are M13 and λ bacteriophage.
Gene cloning 2 b) Vector- Phage/ Phasmid
General pattern of infection
Phage particle attach outside of bacterium cell and injects its DNA.
Phage DNA molecule is replicated inside cell, coding is done by specific number of genes.
Rest genes directs synthesis of capsid, new phage particles are assembled and released from bacterium.
Gene cloning 2 c) Vectors- Cosmid
Circular, extra-chromosomal DNA molecules with combined features of plasmid and phage.
Cloning limit 35-50 times then phage and plasmid.
Gene cloning 2 d) Vectors-BAC
Bacterial artificial chromosome are small pieces of episomal DNA giving conjugation initiating ability to bacteria.
Have cloning limit of 75-300kb.
Gene cloning 2 d) Vectors- YAC
Yeast artificial chromosome replicates in yeast cells.
Have recognition sites for restriction enzymes.
Gene cloning 3. DNA manipulating enzymes
These enzymes are classified in 5 categories.
Nuclease: To cut, shorten, degrade nucleic acids.
a. Exonuclease- can remove one nucleotide at a time from DNA.
b. Endonuclease –Can break internal bonds in DNA molecules
Ligases : Join nucleic acid molecules.
Polymerase : Make copies of molecules.
Modifying enzymes: Remove or add chemical groups.
Topoisomerase : Remove or add supercoils from covalently closed circular DNA.
Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease
Known as DNA scissors.
These are highly specific enzymes that produce internal cuts in DNA.
They act specifically on recognition sites only
Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease- Types
Type-1 Restriction endonuclease e.g. Eco RI
not used in gene cloning.
Type-2 Restriction endonuclease e.g. Hind II, used in gene cloning.
Type-3 Restriction endonuclease e.g. Eco PI
not used in gene cloning.
Gene cloning 3. DNA manipulating enzymes
DNA ligase
Function is to join vector DNA and desired DNA
Purified from t4 phage infected E.Coli bacteria.
Can join blunt ends and sticky ends with later at higher frequency.
Gene cloning 4. Rec. gene transferring modes
Recombinant gene is transferred to host cell by following methods :
Transformation
Transduction
Conjugation
Gene cloning 4. Rec. gene transferring mode
Usually bacterial cell can uptake DNA easily during transformation, but not all species are efficient.
- Electroporation : Giving electric shock of 100-200v and making holes in cell.
- CaCl2 : In presence of Ca 2+ cell wall become permeable to plasmid DNA, heat treatment of 420C for 30-120 sec causes DNA to enter cell.
_ Lipofection : vesicles with desired plasmid is allowed to fuse with cell membrane leading to insertion of gene.
Gene cloning 5. Rec. gene selection and screening mode
Selection : Allowing desired cell to replicate while preventing un- desired cell from replicating.
Screening : Checking clones grown after selection for desired and required properties.
Gene cloning 5. Rec. gene selection and screening mode
Selection and screening techniques:
Antibiotic sensitivity
Enzyme activity
Southern blotting
Colony hybridization
Gene cloning 5. Rec. gene selection and screening mode
Antibiotic sensitivity
If a antibiotic resistance gene is transferred as desired trait, then bacterial colony will grow even if they are treated with antibiotic.
Gene cloning 5. Rec. gene selection and screening mode
Enzyme activity:
Normal cells are able to synthesize β-gal.
Recombinants cannot synthesize by inactivation of β-gal synthesizing gene.
Screening done by X- gal and IPTG.
Recombinant show white color, non-rec shows blue color.
Gene cloning 5. Rec. gene selection and screening mode
Colony Hybridization
Bacteria is transferred from culture plate to nitro-cellulose filter.
Bacteria lysed and DNA denatured.
Incubate and hybridize desired gene with radioactively labeled nucleic acid probe.
Autoradiography is done to expose colony with hybridized probe.
Presented By:Mayank Bhardwaj
Genetic Engineering
Historical Background
Father of Genetics – Gregor John Mendel
- Formulated set of rules to explain inheritance of biological characteristics.
- Basic assumption: Each heritable property is controlled by a factor, Gene.
- Rediscovery of Mendel’s law in 1900 marked birth of Genetics.
- Gene reside on chromosome: W. Sutton (1903)
- Experimental Backing: T.H. Morgan(1910)
- DNA as genetic material: Avery, McCarty, McLeod(1944), Hershey, Chase(1952).
- Between 1952-1966: Structure of DNA proposed, Genetic code cracked, Process of transcription and translation described.
Until decade of 1960 experimental techniques were not sophisticated enough to study gene.
- 1971 to 1973: Revolution in experimental biology.
# Recombinant DNA technology
# Genetic Engineering
Genetic Engineering A Biotechnological Revolution
The synonyms-
Recombinant DNA technology
R-DNA technology
Gene cloning
Molecular cloning
The Applications
Development of GMO and GEO.
Gene therapy (e.g. HGH, Human insulin).
Improving nutritional value of food.
Improving storage life of food.
Controlling water and air pollution.
DNA fingerprinting.
etc..
Gene cloning General process
Fragment of DNA with desired gene inserted to circular DNA molecule (vector) to produce chimaera / r-DNA
Vector act as vehicle for transferring gene to host cell ( gen. bacterium)
Within host cell, vector multiplies producing identical copies. Host cell itself divides and passes r-DNA to progeny.
Copies of desired genes are further extracted by selection and screening process.
Gene cloning Main Steps
Isolation and purification of DNA segments.
Addition of desired gene to vector.
Insertion of recombinant plasmid to host cell.
Selection and screening of clones of r-DNA
Gene cloning Basic Requirements
DNA purification from living cells for desired genes and vectors.
Vectors to carry the desired gene.
DNA manipulating enzymes.
Recombinant Gene transferring modes.
Recombinant Gene selection and screening modes.
Gene cloning 1. DNA purification
Two distinct kind of DNA are purified
a). Total cell DNA: i.e. desired DNA,
donor may be bacteria, animal, plant or any other cell.
Methods used are
- Centrifugation
- Phenol extraction
- Ethanol precipitation
- CTAB method for DNA from plant cell.
Gene cloning 1. DNA purification
B). Vector DNA : vehicle of desired DNA,
Cultures are prepared from plasmid or phages depending upon requirement.
methods used are
- Anion exchange column chromatography
- Alkaline denaturation method
- CsCl density gradient centrifugation
- PEG precipitation ( for phages).
Gene cloning 2. Vectors
It is a DNA molecule carrying foreign DNA to host cell, where it replicates producing its identical copies with foreign DNA.
- Also called Vehicle.
Types of Vector:
Plasmid
Phage/ Phasmid
Cosmid
Yeast artificial chromosome
Bacterial artificial chromosome
Gene cloning 2 a) Vector-Plasmid
Extra-chromosomal, circular DNA in bacteria, having self replicating abilities outside the host cell.
Cloning limit- 0.1 to 10KB.
Gene cloning 2 a) Vector-Plasmid-Types
Classification based on main characteristics coded by plasmid genes.
Fertility (F) plasmid: carry tra genes, have ability for conjugal transfer of plasmid. e.g. F of E.Coli
Resistance ® plasmid: carry gene for antibiotic resistance for host bacterium. e.g. RP4 of Pseudomonas.
Col Plasmid: Colicin code-protein killing other bacteria. E.g. ColE1 of E.Coli.
Degradatice plasmid : allow host bacterium to metabolize unusual molecule as toulene, salicylic acid.
e.g. TOL of Pseudomonas putida.
Gene cloning 2 b) Vector – Phage/ Phasmid
Virus infecting bacteria.
Affect bacteria by lytic and lysogenic cycles.
Although many varieties are there, mainly used are M13 and λ bacteriophage.
Gene cloning 2 b) Vector- Phage/ Phasmid
General pattern of infection
Phage particle attach outside of bacterium cell and injects its DNA.
Phage DNA molecule is replicated inside cell, coding is done by specific number of genes.
Rest genes directs synthesis of capsid, new phage particles are assembled and released from bacterium.
Gene cloning 2 c) Vectors- Cosmid
Circular, extra-chromosomal DNA molecules with combined features of plasmid and phage.
Cloning limit 35-50 times then phage and plasmid.
Gene cloning 2 d) Vectors-BAC
Bacterial artificial chromosome are small pieces of episomal DNA giving conjugation initiating ability to bacteria.
Have cloning limit of 75-300kb.
Gene cloning 2 d) Vectors- YAC
Yeast artificial chromosome replicates in yeast cells.
Have recognition sites for restriction enzymes.
Gene cloning 3. DNA manipulating enzymes
These enzymes are classified in 5 categories.
Nuclease: To cut, shorten, degrade nucleic acids.
a. Exonuclease- can remove one nucleotide at a time from DNA.
b. Endonuclease –Can break internal bonds in DNA molecules
Ligases : Join nucleic acid molecules.
Polymerase : Make copies of molecules.
Modifying enzymes: Remove or add chemical groups.
Topoisomerase : Remove or add supercoils from covalently closed circular DNA.
Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease
Known as DNA scissors.
These are highly specific enzymes that produce internal cuts in DNA.
They act specifically on recognition sites only
Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease- Types
Type-1 Restriction endonuclease e.g. Eco RI
not used in gene cloning.
Type-2 Restriction endonuclease e.g. Hind II, used in gene cloning.
Type-3 Restriction endonuclease e.g. Eco PI
not used in gene cloning.
Gene cloning 3. DNA manipulating enzymes
DNA ligase
Function is to join vector DNA and desired DNA
Purified from t4 phage infected E.Coli bacteria.
Can join blunt ends and sticky ends with later at higher frequency.
Gene cloning 4. Rec. gene transferring modes
Recombinant gene is transferred to host cell by following methods :
Transformation
Transduction
Conjugation
Gene cloning 4. Rec. gene transferring mode
Usually bacterial cell can uptake DNA easily during transformation, but not all species are efficient.
- Electroporation : Giving electric shock of 100-200v and making holes in cell.
- CaCl2 : In presence of Ca 2+ cell wall become permeable to plasmid DNA, heat treatment of 420C for 30-120 sec causes DNA to enter cell.
_ Lipofection : vesicles with desired plasmid is allowed to fuse with cell membrane leading to insertion of gene.
Gene cloning 5. Rec. gene selection and screening mode
Selection : Allowing desired cell to replicate while preventing un- desired cell from replicating.
Screening : Checking clones grown after selection for desired and required properties.
Gene cloning 5. Rec. gene selection and screening mode
Selection and screening techniques:
Antibiotic sensitivity
Enzyme activity
Southern blotting
Colony hybridization
Gene cloning 5. Rec. gene selection and screening mode
Antibiotic sensitivity
If a antibiotic resistance gene is transferred as desired trait, then bacterial colony will grow even if they are treated with antibiotic.
Gene cloning 5. Rec. gene selection and screening mode
Enzyme activity:
Normal cells are able to synthesize β-gal.
Recombinants cannot synthesize by inactivation of β-gal synthesizing gene.
Screening done by X- gal and IPTG.
Recombinant show white color, non-rec shows blue color.
Gene cloning 5. Rec. gene selection and screening mode
Colony Hybridization
Bacteria is transferred from culture plate to nitro-cellulose filter.
Bacteria lysed and DNA denatured.
Incubate and hybridize desired gene with radioactively labeled nucleic acid probe.
Autoradiography is done to expose colony with hybridized probe.
