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Biotechnology
The field of applied biology that involves the use of living things in engineering, technology, medicine, and other useful applications
Introduction
Introduction
Once
these
quality (genes)
traits are
get
Next
Biotechnology
GE of animals GE of plants
GE to improve microorganisms
GE of biocontrol agents against plant pest & diseases Plant protoplast fusion
disease diagnostics
Areas
1.
2.
3.
Areas
4.
Reproductive Biotechnology
5.
Nutritional Biotechnology
Industrial waste use through ruminal fermentation Aflatoxicosis reduction using biomass by yeast Non-conventional feed stuffs
6.
Transgenics Knockouts
Applications in Pakistan
Molecular Characterization
Reproductive Biotechnology Transgenics and Knock outs (GMOs)
DNA structure
DNA sources
DNA 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.
(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
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 Chemistry
Kary Mullis, 1983
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:
1. 2. 3. 4.
5.
6.
PCR Steps
PCR
100
Melting 94 oC
30x
Temperature
50
T i m e
5 3 5 5 3 5
3 5
5 3 5 5 5 3
5 3 5
3
5 3
Temperature
100
Melting 94 oC
PCR
50
T i m e
3 5
5 3
Temperature
100
Melting 94 oC
PCR
50
T i m e
3 5
Heat
5 3
Temperature
100
Melting 94 oC
50
PCR
T i m e
3 5 5
5 5 3
Temperature
PCR
100
Melting 94 oC
50
30x
T i m e
3 5
Heat
5 5
Heat
5 5 3
Temperature
PCR
100
Melting 94 oC
50
30x
T i m e
3
5 5 3
Temperature
PCR
100
Melting 94 oC
50
30x
0
3 5 5
T i m e
5
5
5 3
Heat
5 5
Heat
5
Temperature
PCR
100
Melting 94 oC
50
30x
0
3 5 5
T i m e
5
5
5 3
Temperature
PCR
100
Melting 94 oC
50
30x
0
3 5 5
T i m e
5
5
5 3
16
32
64
0 1 Cycles
If you start with 100 copies, how many copies are made in 30 cycles?
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
DNA
small
large
Power
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
Procedure
Gel tray
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