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DNA EXTREACTION, PCR DNA EXTREACTION, PCR AND AND GEL ELECTROPHORESIS GEL ELECTROPHORESIS

DNA Ext, PCR, Gel Electrophoresis... 30-09-10

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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