09-09-2013, 03:49 PM
Applications of Recombinant DNA Technology
Applications of Recombinant.ppt (Size: 228 KB / Downloads: 33)
Applications
Polymerase Chain Reaction (PCR) has a wide range of applications in many disciplines
Molecular Biology/Research
Diagnostics
Genetic Counseling
Criminology/Forensics
Paternity testing
Archeology
Food testing
Evolutionary studies
Advantages over traditional methodologies
Fast and efficient amplification of specific DNA sequences
No requirement for cloning or subcloning
Tiny amounts of material are usually sufficient
Disease diagnoses will be greatly expedited by PCR to identify microorganisms in infected people who would prove falsely negative by other diagnostic procedures
Drawbacks:
Can introduce errors in DNA during amplification process
Error-prone DNA polymerases
New enzymes are reducing this tendency
Vent polymerase vs. Taq polymerase
Contaminants can give rise to false positives or erroneous results
Numerous controls must be included
Controlled environment
Procedure
A specific DNA sequence is amplified using DNA polymerase and oligonucleotide primers using many cycles (25-30)
~106-fold amplification achieved
Each cycle includes the following steps:
Denaturation (92oC) (separation of dsDNA)
Annealing (55oC) (primer binds to complementary regions)
Extension (72oC) (primer is elongated by DNA polymerase)
Exponential amplification of starting material achieved
Required reagents for PCR:
Template DNA (can be DNA or RNA)
If RNA is used, an extra first step must be introduced involving reverse transcription of RNA to produce DNA template (= Reverse transcription PCR = RT-PCR)
Two flanking oligonucleotide primers (excess)
Nucleotides (excess)
Heat-stable DNA polymerase
Thermocycler to automate process (~6 minutes per cycle, ~3 hours for 30 cycles)
Introduction of foreign genes into intact organisms
Procedure is basically the same regardless of which animal is involved.
Integration usually occurs prior to DNA replication in the fertilized oocyte.
Majority of transgenic animals carry the gene in all of their cells, including the germ cells. Transmission to next generation requires germline integration.
Some integration events occur subsequent to DNA replication giving rise to mosaic animals which may or may not contain the transgene in its germline.
Gene Expression in
Transgenic Mice
In order to discriminate the products of the injected gene from those of an endogenous counterpart, the injected gene must be marked in some way.
Mini-genes where exons are deleted of cDNA where introns are absent.
Modification by insertion/deletion/mutagenesis of a few nucleotides (e.g. the gain or loss of a restriction endonuclease site).
Hybrid genes where foreign epitopes are expressed on transgenic products.
Pluripotent ES Cells
Pluripotent ES cells are undifferentiated early embryonic cells derived from the inner cell mass of mouse blastocysts.
In vitro ES cells must be grown on a feeder layer of fibroblasts to prevent them from differentiating.
Introduction of the transgene is achieved by electroporation of retroviral infection.
The transgene must integrate via recombination, not randomly.
Cells transfected successfully can be identified prior to injection into blastocysts.
Selection of Targeted ES Cells
Gancyclovir resistant and G418 resistant ES cells grow into small clumps on top of feeder cells.
The colonies of cells can be “picked” off and transferred to new wells (at 0.3 cells per well seeding density) containing feeder cells.
When sufficient numbers of cells are obtained, they are:
Frozen for safe storage
Analyzed by Southern blotting or PCR to determine nature of integration event
Microinjected into the blastocoel cavity of blastocysts.