Lecture 1 Introduction to recombinant DNA Technology Dr Muhammad Imran

Lecture 1Introduction 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

1971–1973recombinant 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

PCR: Polymerase Chain Reaction

•Quite different from gene cloning

•Very simple

•Easy to do less time consuming

•Economical

•Wide application

PCR temperature profile

Critical temperature

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

Contents of the reaction

• dNTPs• Tag (enzyme)• Buffer• Primers F and R• Template• MgCl2

• (NH4)2 SO4 or not KCl

PCR reaction contents

https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction

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)

Continued………………..Parameters Optimum Comments

3' End Stability

Avoid Template Secondary Structure

Avoid Cross Homology

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?

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/

Degenerate Primers• When the polymorphism in region to be

amplified exist.• When primers have to be designed from

protein sequence or conserved protein domain

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

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’

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

ARMS (Amplification refractory mutation system)

Watch out, outer primer colors are bright and light you may have difficulty in seeing them

Primers for protein expression

• 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

• 5'-extension to the restriction site

http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/extensions.pdf

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

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

Primers for site directed mutagenesis

• 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

http://oxfordgenetics.com/cloning-resources/cloning-guides/site-directed-mutagenesis