Biotechnology

DR. MD.MAHBUBUR RAHMAN MBBS, MPhil. MSc. (Biotechnology)

Assistant Professor Dept. of Biochemistry

RAJSHAHI MEDICAL COLLEGE

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

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.

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.

Recombinant DNA

Recombinant DNA refers to techniques that are used to manipulate, move, recombine and propagate DNA.

Continued .......

The potential use of these techniques for the diagnosis and treatment of disease are vast.

Tools of recombinant DNA

• Restriction endonuclease that permit the dissection of huge DNA molecule into defined fragment.

Continued........... • Other nucleic acid enzyme

oDNA polymerase

oRNA dependent DNA polymerase.

Use of recombinant DNA techniques

• DNA Polymorphisms

• Detection of polymorphism

Continued

The prevention and treatment of diseases.

Vaccine

Production of therapeutic protein

eg Insulin, Growth Hormone

Complex human protein –Factor VIII, TPA, IL.

DNA Cloning

• A clone refers to the cell with an identical genotype.

• Amplification of DNA is known as cloning.

VECTOR • A vector is molecule of

DNA to which the fragment of DNA is attached.

Properties of vector

oAutonomous replication

oCapable of insertion into host cell

oMust contain one specific nucleotide recognized by restriction endonuclease.

Types of vector

•Plasmid , Cosmid,

•Bacteriophages

•YAC

Function of Vector

POLYMERASE CHAIN REACTION

• Is the test tube method for amplification of selected DNA to large number of identical copies.

Continued......

• PCR is an vitro method that can be used for rapid production of very large amount of specific segments of DNA.

STEPS OF PCR • Primer construction

• Denature the DNA

• Annealing of primers to single-stranded DNA

• Chain extension

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

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.

Probes • Single stranded pieces of DNA usually labeled

with radioisotope such as 32P.

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

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.

Complementary DNA library

Contains only those DNA sequence that appear as mRNA molecules.

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.

DNA variation resulting in RFLP

• Single base change in DNA

• Tandem repeats ( VNTR)

Prenatal Diagnosis

• Source of DNA

oFrom white blood cell

oamniotic fluid

oChorionic villi.

Blotting techniques

• Southern Blot --- Analysis of DNA

• Northern Blot --- Analysis of RNA

• Western Blot ----Analysis of Protein