Public Transport in Pakistan: A Critical Overview by Muhammad Imran
Lecture 1 Introduction to recombinant DNA Technology Dr Muhammad Imran.
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Transcript of Lecture 1 Introduction to recombinant DNA Technology Dr Muhammad Imran.
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Lecture 1Introduction to recombinant DNA Technology
Dr Muhammad Imran
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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.
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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
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History of rDNA Technology
Gregor Mendel1850s and 1860s the birth of genetics
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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
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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
1971–1973recombinant DNA technology or genetic engineering
Gene cloning
Kary Mullis discovered a revolutionary technique now called PCR
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Gene Cloning
T.A Brown 6th Edition
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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
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PCR: Polymerase Chain Reaction
•Quite different from gene cloning
•Very simple
•Easy to do less time consuming
•Economical
•Wide application
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PCR temperature profile
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Critical temperature
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Melting temperature or Tm of Primers
Melting Temperature or Tm. The Tm is the temperature at which the correctly base-paired hybrid dissociates (“melts”).
Tm = (4 × [G + C]) + (2 × [A + T])°C
TAB
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Contents of the reaction
• dNTPs• Tag (enzyme)• Buffer• Primers F and R• Template• MgCl2
• (NH4)2 SO4 or not KCl
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PCR reaction contents
https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction
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Principle of Primer designing• Few things to be considered while designing the primers
Parameters Optimum Comments
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 StructuresRepeats 4 dinucleotide repears allowed eg ATATATAT
Runs Consecutive single nucleotide repeat of 4 max allowed (otherwise mispriming)
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Continued………………..Parameters Optimum Comments
3' End Stability
Avoid Template Secondary Structure
Avoid Cross Homology
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Primer for different purposes
• Simple primer (Universal)• Degenerate primers• ARMS PCR Primer• Multiplex PCR primers• Primers for protein expression• Primers for site directed
mutagenesis
• When we need them?
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Simple Primer (universal primers)• When we want to amplify a region for sequencing,
homologous sequences are available to design primers in large number.
• 16S rDNA primers (Universal primer)• When large data of identical sequences is known ClCuD universal primers • Universal primers for sequencing clones in expression
vectors or TA cloning vectors etc.T7 promoter forward: TAATACGACTCACTATAGGGT7 terminator reverse: GCTAGTTATTGCTCAGCGG
http://www.addgene.org/mol_bio_reference/sequencing_primers/
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Degenerate Primers• When the polymorphism in region to be
amplified exist.• When primers have to be designed from
protein sequence or conserved protein domain
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A C G T A/C A/g A/T C/g C/T g/T A/C/g A/C/T A/g/T C/g/T A/C/g/T
A C G T M R W S Y K V H D B N
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Degenerate primers cont……141
A C G T A/C A/g A/T C/g C/T g/T A/C/g A/C/T A/g/T C/g/T A/C/g/T
A C G T M R W S Y K V H D B N
F Primer 5’ ACN gAR gCN CAR TAY gAR gAR ATg 3’
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Reverse degenerate primer 233
A C G T A/C A/g A/T C/g C/T g/T A/C/g A/C/T A/g/T C/g/T A/C/g/T
A C G T M R W S Y K V H D B N
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ARMS (Amplification refractory mutation system)
Watch out, outer primer colors are bright and light you may have difficulty in seeing them
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Primers for protein expression
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• Restriction site. Should be same as for vector or produce same sticky ends as of 1st site in MCS Blunt end cutter Nco I CCATGG) or Nde I (CATATG) are mostly
chosen as the ATG within these sites can be used directly to create the ATG start codon and/or the ATG codon for the N-terminal methionine residue.
Design of the 5'-end primerFollowing modification can be made to the 5 prime
end of a primer
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• 5'-extension to the restriction site
http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/extensions.pdf
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Design of the 5'-end primer
• Start codon. 1) If no N terminal tag/fusion partner….include a
start codon (usually ATG). 2)- or if an N-terminal methionine residue is
present. 3) Make sure that N terminal tag/fusion partner is
in frame with gene of interest• Overlap with the gene of interest. The overlap
between the primer and the gene of interest should be long enough to give a Tm of 60°C or more
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Design of the 3'-end primer• Restriction site. Should be same as for vector or produce same sticky ends as of 2nd or 3rd site in MCS or use blunt end cutter• Stop codon(s). If no C-terminal tag introduce a stop codon TAA is preferred over TAG/TGA Two or three stop codon sequences can be
incorporated for efficient termination• Overlap with the gene of interest. The overlap between the primer and the gene of
interest should be long enough to give a Tm of 60°C or more
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Primers for site directed mutagenesis
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• Back to back primer design (principle)
• Incorporate the desired mutation at the 5 end of a primer or both primers
• We usually insert the mutation in the middle of the primer (better result, draw on board)
• Calculate the Tm considering mismatches
http://depts.washington.edu/bakerpg/primertemp/primertemp.html
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http://oxfordgenetics.com/cloning-resources/cloning-guides/site-directed-mutagenesis
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