Download Lecture 1 Introduction to recombinant DNA Technology

Lecture 1
Introduction to recombinant DNA
Technology
Dr Muhammad Imran
What is a gene?
• Gene is a piece of DNA which encode an RNA
molecule which may encode a protein
What is a genetic engineering?
• Set of techniques by which one can deliberately
insert new piece/s of DNA into the existing DNA
piece to modify the characters of an organism.
Gene Cloning
• Set of experiments carried out to create a
recombinant molecule and its propagation in an
organism/host organism multiplication.
PCR: Polymerase Chain Reaction
• A reaction in which we use DNA polymerase
to make the copies of fragment of DNA
selectively amplified with the help of primers
History of rDNA Technology
Gregor Mendel1850s and 1860s the birth of genetics
What genes are and how they work
• W. Sutton…the factors (genes) reside on
Chromosomes 1903.
• TH Morgan ……. Endorsed Sutton…..and gene
mapping started in 1910 and by 1922 nearly
2000 genes were mapped.
• Set of experiments by Avery, MacLeod, and
McCarty in 1944, and of Hershey and Chase
in 1952 proved that DNA is hereditary
material and not the proteins
1952-1966 well-done Watson and
Crick
• Structure of DNA was elucidated, genetic code
cracked, and the processes of
• transcription and translation described
Anticlimax era and frustration in late 1960
1971–1973
recombinant DNA technology or genetic engineering
Gene cloning
Kary Mullis discovered a revolutionary technique
now called PCR
Gene Cloning
T.A Brown 6th Edition
Properties to DNA and its replication
• DNA is double helix
• Double helix is anti-parallel
• Replication only takes place from 5-3
• Replication is semi conservative
• Replication is bidirectional
•Quite different from gene cloning
•Very simple
•Easy to do less time consuming
•Economical
•Wide application
PCR: Polymerase Chain Reaction
Principle of Primer designing
• Few things to be considered while designing the primers
Parameters
Optimum
Primer Length
18-22
Primer Melting
Temperature
52-58 oC
Primer Annealing
Temperature
Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9
GC Content
40-60%
GC Clamp
Primer Secondary
Structures
Repeats
4 dinucleotide repears allowed eg ATATATAT
Runs
Consecutive single nucleotide repeat of 4
max allowed (otherwise mispriming)
Comments
Continued………………..
Parameters
3' End Stability
Avoid Template Secondary Structure
Avoid Cross Homology
Optimum
Comments