biotechnology - rmc.gov.bdrmc.gov.bd/notice_panel/upload_notices/recombinant_dna.pdf ·...
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Biotechnology
DR. MD.MAHBUBUR RAHMAN MBBS, MPhil. MSc. (Biotechnology)
Assistant Professor Dept. of Biochemistry
RAJSHAHI MEDICAL COLLEGE
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At the end of session student will be able to
Definition of Biotechnology
Definition of Vector & it’s criteria.
Definition of Clone, Probe, DNA library.
Definition of PCR, Steps and application
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Definition of BT &MBT
• Biotechnology is the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life.
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Continued........
Biotechnology was achieved in the Convention on Biological Diversity (1992) – "any technological application that uses biological systems, living organisms or derivatives there of, to make or modify products and processes for specific use.
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Recombinant DNA
Recombinant DNA refers to techniques that are used to manipulate, move, recombine and propagate DNA.
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Continued .......
The potential use of these techniques for the diagnosis and treatment of disease are vast.
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Tools of recombinant DNA
• Restriction endonuclease that permit the dissection of huge DNA molecule into defined fragment.
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Continued........... • Other nucleic acid enzyme
oDNA polymerase
oRNA dependent DNA polymerase.
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Use of recombinant DNA techniques
• DNA Polymorphisms
• Detection of polymorphism
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Continued
The prevention and treatment of diseases.
Vaccine
Production of therapeutic protein
eg Insulin, Growth Hormone
Complex human protein –Factor VIII, TPA, IL.
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DNA Cloning
• A clone refers to the cell with an identical genotype.
• Amplification of DNA is known as cloning.
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VECTOR • A vector is molecule of
DNA to which the fragment of DNA is attached.
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Properties of vector
oAutonomous replication
oCapable of insertion into host cell
oMust contain one specific nucleotide recognized by restriction endonuclease.
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Types of vector
•Plasmid , Cosmid,
•Bacteriophages
•YAC
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Function of Vector
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POLYMERASE CHAIN REACTION
• Is the test tube method for amplification of selected DNA to large number of identical copies.
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Continued......
• PCR is an vitro method that can be used for rapid production of very large amount of specific segments of DNA.
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STEPS OF PCR • Primer construction
• Denature the DNA
• Annealing of primers to single-stranded DNA
• Chain extension
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Application of PCR
• Comparison of normal cloned gene with an uncloned mutant form of the gene
• Detection of low abundance nucleic acid sequence.
• Forensic analysis of DNA samples
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Advantage of PCR The major advantage of PCR over cloning are sensitivity and speed.
•DNA sequence present in the individual cell can be amplified and studied.
•Isolating and amplifying of a specific DNA sequence by PCR is faster and easier than traditional cloning methods.
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Probes • Single stranded pieces of DNA usually labeled
with radioisotope such as 32P.
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DNA Library
Is a collection of cloned restriction fragments of the DNA of an organism.
Types of DNA Library
Genomic DNA libraries
Complementary DNA libraries
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Genomic DNA libraries
• Is the collection of fragments of double stranded DNA
obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector.
OR
• Refers to a bank or library of clones that contains every sequence from the genome of a specific organism.
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Complementary DNA library
Contains only those DNA sequence that appear as mRNA molecules.
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RFLP • Is a technique in which restriction fragment
markers that demonstrate tight linkage to a mutant phenotype are identified.
• A polymorphic gene is one in which the variant alleles are common enough to be useful as genetic markers usually 1% or more.
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DNA variation resulting in RFLP
• Single base change in DNA
• Tandem repeats ( VNTR)
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Prenatal Diagnosis
• Source of DNA
oFrom white blood cell
oamniotic fluid
oChorionic villi.
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Blotting techniques
• Southern Blot --- Analysis of DNA
• Northern Blot --- Analysis of RNA
• Western Blot ----Analysis of Protein