DNA EXTREACTION, PCR DNA EXTREACTION, PCR AND AND
GEL ELECTROPHORESISGEL ELECTROPHORESIS
The field of applied biology that involves the use of living things in engineering, technology,
medicine, and other useful applications
Biotechnology
Introduction
Biotechnology today is the mother of all Biological Biotechnology today is the mother of all Biological Sciences.Sciences.
There is hardly any area in Biology that has not been There is hardly any area in Biology that has not been touched by Biotechnology. touched by Biotechnology.
It identifies DNA markers associated with disease It identifies DNA markers associated with disease resistance, milk and meat production traits.resistance, milk and meat production traits.
It offers exceptionally powerful alternatives to classical It offers exceptionally powerful alternatives to classical genetics in determining linkage analysis of traits.genetics in determining linkage analysis of traits.
Next……
Introduction
Once these quality traits get Once these quality traits get
established and their respective DNA established and their respective DNA
sequences (genes) are known, sequences (genes) are known,
recombinant DNA technology will be recombinant DNA technology will be
put to use for producing genetically put to use for producing genetically
modified animals, plants, medicines modified animals, plants, medicines
etc.etc.
Next……
GE of animalsGE of animals
GE to develop animal vaccinesGE to develop animal vaccines
GE of biocontrol agents against plant pest & diseases
GE of biocontrol agents against plant pest & diseases
Plant protoplast fusionPlant protoplast fusion
Embryo transferEmbryo transfer
GE of plantsGE of plants
GE to improve microorganismsGE to improve microorganisms
Recombinant DNA for disease diagnosticsRecombinant DNA for disease diagnostics
Monoclonal anti body productionMonoclonal anti body production
Plant tissue culturePlant tissue culture
Fermentation, BiofertilizersFermentation, Biofertilizers
Biotechnology
Areas1.1. Molecular Characterization of Animals and MicrobesMolecular Characterization of Animals and Microbes
DNA fingerprinting and genetic markersDNA fingerprinting and genetic markers
Gene Sequencing and genome mappingGene Sequencing and genome mapping
DNA Bank of Native breeds and strainsDNA Bank of Native breeds and strains
2.2. Recombinant DNA technologiesRecombinant DNA technologies Biotherapeutics technology for vaccines and medicinesBiotherapeutics technology for vaccines and medicines
3.3. Recombinant Protein Production and PurificationRecombinant Protein Production and Purification Genetic EngineeringGenetic Engineering
Protein PurificationProtein Purification
Diagnostic Protein KitsDiagnostic Protein Kits
Next……
Areas
4.4. Reproductive BiotechnologyReproductive Biotechnology AI, IVF, IVM, ETT, MOET and CloningAI, IVF, IVM, ETT, MOET and Cloning
5.5. Nutritional BiotechnologyNutritional Biotechnology
Industrial waste use through ruminal fermentationIndustrial waste use through ruminal fermentation
Aflatoxicosis reduction using biomass by yeastAflatoxicosis reduction using biomass by yeast
Non-conventional feed stuffsNon-conventional feed stuffs
6.6. Genetically Modified Organisms (GMOs)Genetically Modified Organisms (GMOs)
TransgenicsTransgenics
Knockouts Knockouts
Applications in Pakistan
Molecular Characterization Molecular Characterization
Reproductive BiotechnologyReproductive Biotechnology
Transgenics and Knock outs (GMOs)Transgenics and Knock outs (GMOs)
Health Care (therapeutics & diagnostics)Health Care (therapeutics & diagnostics)
Recombinant DNA Vaccine ProductionRecombinant DNA Vaccine Production
Recombinant Protein Production Recombinant Protein Production
Cell Culture SystemsCell Culture Systems
Let’s Start with DNALet’s Start with DNA
Founder of DNA StructureFounder of DNA Structure
Watson & Crick 1953
DNA: DNA: The Molecule of lifeThe Molecule of life
DNA structureDNA structure
DNA sourcesDNA sources
DNA can be isolated from any nucleated cell.DNA can be isolated from any nucleated cell. BloodBlood Buccal cellsBuccal cells Cultured cellsCultured cells Bacterial plasmids, cosmidsBacterial plasmids, cosmids BiopsiesBiopsies Forensic samples i.e. body fluids, hair follicles, bone Forensic samples i.e. body fluids, hair follicles, bone
& teeth roots. & teeth roots.
The Standard Principle of DNA IsolationThe Standard Principle of DNA Isolation
(1)(1) Lysis of cellsLysis of cells::
Lysis buffer: SDS and/or 8.0 M ureaLysis buffer: SDS and/or 8.0 M urea (2) Removal of contaminants:(2) Removal of contaminants:
Proteinase KProteinase K
Phenol: chloroform extractionPhenol: chloroform extraction (3) Concentration of DNA:(3) Concentration of DNA:
Ethanol/Isopropanol precipitationEthanol/Isopropanol precipitation
Polymerase Chain ReactionPolymerase Chain Reaction (PCR) (PCR)
HistoryHistory
The Polymerase Chain Reaction (PCR) was not a The Polymerase Chain Reaction (PCR) was not a discovery, but rather an inventiondiscovery, but rather an invention
A special DNA polymerase (A special DNA polymerase (TaqTaq) is used to make ) is used to make many copies of a short length of DNA (100-10,000 many copies of a short length of DNA (100-10,000 bp) defined by primersbp) defined by primers
Kary Mullis, the inventor of PCR, was awarded the Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry1993 Nobel Prize in Chemistry
Kary Mullis, 1983
How PCR WorksHow PCR Works
PCR is an artificial way of doing DNA replicationPCR is an artificial way of doing DNA replication
Instead of replicating all the DNA present, only a small Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated segment is replicated, but this small segment is replicated many timesmany times
As in replication, PCR involves:As in replication, PCR involves: Melting DNAMelting DNA PrimingPriming Polymerization Polymerization
Components of PCR ReactionComponents of PCR Reaction
1. Template DNA
2. Buffer
3. 2 Primers
4. dNTPs
5. Taq DNA Polymerase
6. Water
PCR StepsPCR Steps
PCRPCRMelting
94 oC
Melting
94 oC
AnnealingPrimers
50 oC
Extension
72 oCT
empe
ratu
re
100
0
50
T i m e
30x
5’3’
3’5’
3’5’
5’
5’3’5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
PCRPCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
Fragments of defined length
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
DNA Between The Primers Doubles DNA Between The Primers Doubles With Each Thermal CycleWith Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
Theoretical Yield of PCRTheoretical Yield of PCRTheoretical yield = 2n x y
Where y = the starting number of copies and
n = the number of thermal cycles
= 107,374,182,400
If you start with 100 copies, how many copies are made in 30 cycles?
2n x y
= 230 x 100
= 1,073,741,824 x 100
GEL ELECTROPHORESISGEL ELECTROPHORESIS
Gel electrophoresis separates molecules on the basis Gel electrophoresis separates molecules on the basis of their charge and size. of their charge and size.
The charged macromolecules migrate across a span of The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel because they are placed in an electrical field. The gel acts as a sieve to to retard the passage of gel acts as a sieve to to retard the passage of
molecules according to their size and shape.molecules according to their size and shape.
• DNA is negatively charged.
+-
Power
DNA
• When placed in an electrical field, DNA will migrate toward the positive pole (anode).
H
O2
• An agarose gel is used to slow the movement of DNA and separate by size
Scanning Electron Micrograph
of Agarose Gel (1×1 µm)
• Polymerized agarose is porous,
allowing for the movement of DNA
+-
Power
DNA
How fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!*Small DNA move faster than large DNA…gel electrophoresis separates DNA according to size
smalllarge
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
ProcedureProcedure
Gel trayGel tray
Gel Comb…different sizesGel Comb…different sizes
Pouring of Gel into Gel TrayPouring of Gel into Gel Tray
Buffer solution added to the tankBuffer solution added to the tank
DNA samples loading into wellsDNA samples loading into wells
Electrical current applied gel apparatusElectrical current applied gel apparatus
Gel viewed on UV IlluminatorGel viewed on UV Illuminator
Gel Documentation SystemGel Documentation System
DNA bands by Gel Doc systemDNA bands by Gel Doc system
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