Author
moisil-marya
View
14
Download
1
Tags:
Embed Size (px)
DNA EXTREACTION, PCR AND GEL ELECTROPHORESIS
BiotechnologyThe field of applied biology that involves the use of living things in engineering, technology, medicine, and other useful applications
Introduction
Biotechnology today is the mother of all Biological Sciences.There is hardly any area in Biology that has not been touched by Biotechnology. It identifies DNA markers associated with disease resistance, milk and meat production traits. It offers exceptionally powerful alternatives to classical genetics in determining linkage analysis of traits.Next
Introduction
Once
these
quality (genes)
traits are
get
established and their respective DNAsequences known, recombinant DNA technology will be
put to use for producing geneticallymodified animals, plants, medicines etc.
Next
BiotechnologyGE of animals GE of plants
GE to develop animal vaccines
GE to improve microorganisms
Recombinant DNA for
GE of biocontrol agents against plant pest & diseases Plant protoplast fusion
disease diagnostics
Monoclonal anti body production Plant tissue culture
Embryo transfer Fermentation, Biofertilizers
Areas1.
Molecular Characterization of Animals and Microbes
DNA fingerprinting and genetic markersGene Sequencing and genome mapping DNA Bank of Native breeds and strains
2.
Recombinant DNA technologies
Biotherapeutics technology for vaccines and medicines
3.
Recombinant Protein Production and Purification
Genetic Engineering Protein Purification Diagnostic Protein KitsNext
Areas4.
Reproductive Biotechnology
AI, IVF, IVM, ETT, MOET and Cloning
5.
Nutritional Biotechnology
Industrial waste use through ruminal fermentation Aflatoxicosis reduction using biomass by yeast Non-conventional feed stuffs
6.
Genetically Modified Organisms (GMOs)
Transgenics Knockouts
Applications in Pakistan
Molecular CharacterizationReproductive Biotechnology Transgenics and Knock outs (GMOs)
Health Care (therapeutics & diagnostics)Recombinant DNA Vaccine Production Recombinant Protein Production
Cell Culture Systems
Lets Start with DNA
Founder of DNA Structure
Watson & Crick 1953
DNA: The Molecule of life
DNA structure
DNA sourcesDNA can be isolated from any nucleated cell. Blood Buccal cells Cultured cells Bacterial plasmids, cosmids Biopsies Forensic samples i.e. body fluids, hair follicles, bone & teeth roots.
The Standard Principle of DNA Isolation
(1) Lysis of cells: Lysis buffer: SDS and/or 8.0 M urea (2) Removal of contaminants: Proteinase K Phenol: chloroform extraction (3) Concentration of DNA: Ethanol/Isopropanol precipitation
Polymerase Chain Reaction (PCR)
History
The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in ChemistryKary Mullis, 1983
How PCR Works
PCR is an artificial way of doing DNA replication Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times As in replication, PCR involves:
Melting DNA Priming Polymerization
Components of PCR Reaction
1. 2. 3. 4.
Template DNA Buffer 2 Primers dNTPs
5.6.
Taq DNA PolymeraseWater
PCR Steps
PCR100
Melting 94 oC Annealing Primers 50 oC Extension 72 oC
Melting 94 oC
30x
Temperature
50
0
T i m e5 3 5 5 3 5
3
5
3
5
5
3 5
5 3 5 5 5 3
5 3 5
5
35 3
Temperature
100
Melting 94 oC
PCR
50
0
T i m e
3 5
5 3
Temperature
100
Melting 94 oC
PCR
50
0
T i m e3 5
Heat5 3
Temperature
100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
PCR
0
T i m e3 5 5
5 5 3
Temperature
PCR100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
30x
0
T i m e3 5
Heat5 5
Heat5 5 3
Temperature
PCR100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
30x
0
T i m e3
5
5
5
5
5
5
5 5 3
Temperature
PCR100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
30x
03 5 5
T i m e5
55 3
Heat5 5
Heat5
Temperature
PCR100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
30x
03 5 5
T i m e5
5
55 3
5
5
5
5
5
5
Temperature
PCR100
Melting 94 oC
50
Melting o Extension 94 C Annealing 72 oC Primers 50 oC
30x
03 5 5
T i m e5
5
55 3
Fragments of defined length
5
5
5
5
5
5
DNA Between The Primers Doubles With Each Thermal CycleNumber 1 2
4
8
16
32
64
0 1 Cycles
2
3
4
5
6
Theoretical Yield of PCRTheoretical yield = 2n x yWhere y = the starting number of copies and n = the number of thermal cycles
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 = 107,374,182,400
GEL ELECTROPHORESIS
Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to to retard the passage of molecules according to their size and shape.
DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size
H DNA
O2
-
+
Power
Polymerized agarose is porous, allowing for the movement of DNAScanning Electron Micrograph of Agarose Gel (11 m)
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
DNA
smalllarge
-
+
Power
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
Procedure
Gel tray
Gel Combdifferent sizes
Pouring of Gel into Gel Tray
Buffer solution added to the tank
DNA samples loading into wells
Electrical current applied gel apparatus
Gel viewed on UV Illuminator
Gel Documentation System
DNA bands by Gel Doc system
Thank You