Chapter 4

Recombinant DNA Technology Recombinant DNA Technology

 Generation of recombinant DNA molecule  Cloning vector-insert DNA construct (DNA construct)  Cut DNA from a donor organism  Cloned DNA, insert DNA, target DNA, foreign DNA  Ligation to a cloning vector DNA

 Transformation  Introduction and maintain the DNA construct within a host cell

 Selection of transformed cells

 Production of the foreign protein in the host (optional) Recombinant DNA Technology DNA Cloning

Preparation of Insert DNA Genomic cDNA PCR DNA

Joining to a vector

Ligation Recombination

E. coli transformation and selection 1. Cutting DNA Molecules Cutting DNA Molecules

 Mechanical shearing

 Discovery of endonuclease  Meselson and Yuan, 1968, from E. coli  Smith and Wilcox, 1970 from Haemophilus influenzae  Werner Arber, Daniel Nathans and Hamilton Smith :Nobel Prize for physiology or Medicine, 1978 Restriction and Modification

 Bacterial protection fd system against Invasion of foreign E. coli K DNA  Phage fd K fd K  Modified during growth in E.coli K E. coli K E. coli B  Restricted in growth in E.coli B Restriction and Modification

Restriction: Endonuclease

Modification: Methylase Types of Endonuclease System

Recognition Cleavage System Enzyme Subunits sites sites

3 (Recognition, Type I 1 Methylation, No feature Up to 1 kb away Cleavage)

2 (Cleavage, Type II Symmetrical Recognition site Modification) 2 (Recognition and Type III 1 Modification, Symmetrical 24-26 bp away Cleavage) Up to 20 bp 2 (Cleavage, Type IIs Asymmetrical away Modification) Advantage of Type II systems

 Separate restriction and modification : Cleavage without modification

 No cofactors necessary for restriction activity

 Recognize defined sequence (symmetrical, palindromic sequence)

 Cut within the recognition sequence Nomenclature

Sma I 1st enzyme

Serratia marcescencs : species

Hind III 3rd enzyme

Strain d Haemophilus influenzae : species Cleavage by EcoRI

 Recognition site: GAATTC  Symmetrical staggered cleavage  5’ overhang, protruding ends, sticky ends  5’ phosphate and 3’ hydroxyl group Cleavage by HindII

 Recognition site: GTTAAC  Blund-end cleavage Restriction Patterns

 Cohesive ends  5’ overhang: Major, EcoRI, BamHI, etc.

5’ G/AATTC 3’ 5’ G AATTC 3’ 3’ CTTAA/G 5’ 3’ CTTAA G 5’

 3’ overhang: PstI, KpnI

5’ CTGCA/G 3’ 5’ CTGCA G 3’ 3’ G/ACGTC 3’ G ACGTC 5’

 Blunt ends  SmaI, EcoRV 5’ CCC/GGG 3’ 5’ CCC GGG 3’ 3’ GGG/CCC 5’ 3’ GGG CCC 5 Recognition Sequences

 >5000 enzymes  http://rebase.neb.com/rebase/rebase.html

 4-Base Cutters  DpnI/Sau3AI, AluI

 6-Base Cutters  EcoRI, BglII, PvuII

 8-Base Cutters  NotI, SbfI Restriction Enzymes

 Isoschizomer  Enzymes that recognize the same target DNA sequence and cleave it in the same way  e.g. SphI and BbuI (CGTAC/G)

 Enzymes that recognizes the same target DNA sequence but cleave at different points  e.g. SmaI (CCC/GGG) and XmaI (C/CCGGG)

 Isocaudomers  Enzymes that produce the same nucleotide extensions but have different recognition sites  e.g. BamHI (G/GATCC) and Sau3AI (/GATC) Methylases in E. coli

Frequency Methylase Recognition Sequence (if 50% GC)

dam GAmTC (N6) 256 bp

dcm CmCA/TGG (C5) 512 bp

M.EcoKI AAmCGTGC GCAmCGTT (N6) 8 kb

 Restriction enzymes have different preference for methylated DNA  e.g. MboI (GATC), DpnI (GAmTC), Sau3AI (GAmTC, GATC) EcoRII (CCA/TGG), BstNI (CmCA/TGG, CCA/TGG)

 Reduced transformation efficiency of methylated DNA to other species: use dam-, dcm- strain for DNA preparation 2. Separation of DNA Molecules Gel Electrophoresis .  Electrophoresis  A technique used to separate macromolecules (proteins and nucleic acids) that differ in size, charge or conformation

 Migration of molecules in an electric field  DNA (negative charge): migrate toward positive pole  Protein: migrate either positive or negative pole according to their charge  SDS PAGE: proteins are treated with sodium dodecyl sulfate (SDS)  Similar charge to mass ratio  Migration according to the molecular weight Types of Gel

 Agarose  Polysaccharide extracted from seaweed  0.5 to 2%  Used for DNA and RNA  Large range of separation (0.1 to 50 kb DNA)  Low resolving power

 Polyacrylamide  Cross-linked polymer of acrylamide  3.5 to 20%.  Used for DNA, RNA, and protein  Small range of separation (<500 bp DNA)  High resolving power  Inhibition of polymerization process by oxygen  Neurotoxin Agarose Gel Electrophoresis Migration of DNA in Agarose Gel

 Molecular weight of DNA  Conformation of DNA  Supercoil > Linear> Nicked circle  Agarose Concentration  Higher concentration : better separation of smaller DNAs  low concentrations : better resolution of larger DNAs

Migration distance

Log10 MW Migration of DNA in Agarose Gel (2)

 Voltage  High voltage  Lower resolution of large DNA  For the resolution of DNA larger than 2 kb  <5 volts/cm (between two electrode)  Electrophoresis buffer  TAE (Tris-acetate-EDTA), TBE (Tris-borate-EDTA)  Provide ions to support conductivity  Establish pH Ethidium Bromide

 A fluorescent dye that intercalates between bases of nucleic acids Restriction Mapping of DNA

 Cut DNA with various endonuclease  Determination of the sizes of the restriction fragments by gel electrophoresis 3. Enzymes for Recombinant DNA Technology DNA 5’ End labeling 1

 Calf intestine alkaline phosphatase  Dephosphorylation of 5’ end  T4 polynucleotide kinase  Addition of radioisotope-labeled g32P-ATP g-phosphate from g- 32P ATP DNA 5’ End labeling 2

 Filling in reaction with Klenow fragment

a32P-dATP Enzymes Used for Recombinant DNA Technology

 Alkaline phosphatase

 DNaseI  Digestion of dsDNA

 E. coli exonulcease III  Digestion from recessive or blunt 3’ OH ends

 Klenow fragment  E.coli DNA polymerase I with polymerase and 3’ exonuclease activity

 Mung bean nuclease  Digestion of ssDNA and RNA Enzymes Used for Recombinant DNA Technology

 Poly(A) polymerase  Addition of AMP to the 3’ end of mRNA

 Reverse transcriptase  Synthesis of DNA from RNA

 RNaseH  Degrades the RNA strand from a DNA-RNA hybrid

 S1 nuclease  Digestion of ssDNA 4. Joining DNA Molecules Cloning DNA

 Annealing of cohesive ends by base-pairing  Generation of nick Variations on Cutting and Joining DNA

 Compatible cohesive ends 5’ A/CCGGT 3’ AgeI 3’ TGGCC/A 5’ + + 5’ A/CCGGT 3’ 5’ C/CCGGG 3’ AgeI AvaI 3’ TGGCC/A 5’ 3’ GGGCC/C5’

5’ A/CCGGT 3’ 5’ A/CCGGT 3 5’ A/CCGGG 3’ 5’ C/CCGGT 3’ 3’ TGGCC/A 5’ 3’ TGGCC/A 5 3’ TGGCC/C 5’ 3’ GGGCC/A5’

 Blunt ends 5’ CCC/GGG 3’ 5’ GAT/ATC 3’ SmaI EcoRV 3’ GGG/CCC 5’ + 3’ CTA/TAG5

5’ CCC/ATC 3’ 5’ GAT/GGG 3’ 3’ GGG/TAG 5’ 3’ CTA/CCC5’ DNA ligase

 Formation of phosphodiester bonds between 3’ OH and 5’ phosphate

T4 DNA ligase E. coli ligase Ligation Conditions

 Temperature  Consider enzyme activity and base pairing of cohesive termini  Cohesive ends: 4-15oC: ensure base pairing  Blunt ends: 18oC, use 10 to 100 times higher concentration of T4 DNA ligase

 DNA concentration  Dilute concentration favors circulization of linear fragment  Insert : Vector = 2 : 1 molar ratio

 Phosphatase treatment  Prevention of self ligation of vector Ligation Strategy

 Linker linker  Blunt ends DNA containing site RE  Adaptor  Chemically synthesized DNA with cohesive ends adaptor  Cloning of PCR Products  Pfu polymerase  Blunt end ligation  Taq polymerase:  Blunt end ligation after filling in with Klenow  Use T/A cloning with a vector containing 3’ T  Addition of restriction enzyme sites at the end of primers  Add additional 3-4 nucleotide for efficient cleavage R1 PCR 5’ 3’ 3’ 5’

R2 R1 R2 Efficiency of Enzyme Digestion

% Cleavage

Enzyme Oligo Sequence Chain length 2 hr 20 hr

CGGATCCG 8 10 25 BamHI CGGGATCCCG 10 >90 >90 CGCGGATCCGCG 12 >90 >90

GGAATTCC 8 >90 >90 EcoRI CGGAATTCCG 10 >90 >90 CCGGAATTCCGG 12 >90 >90

CAAGCTTG 8 0 0 HindIII CCAAGCTTGG 10 0 0 CCCAAGCTTGGG 12 10 75

CCCGGG 6 0 10 CCCCGGGG 8 0 10 Sma I CCCCCGGGGG 10 10 50 TCCCCCGGGGGA 12 >90 >90

CTCTAGAG 8 0 0 GCTCTAGAGC 10 >90 >90 Xba I TGCTCTAGAGCA 12 75 >90 CTAGTCTAGACTAG 14 75 >90 Cloning Using in vitro Recombination

 Vector and insert with recognition sites for site- specific recombinase  l integrase  Flp recombinase

Recombinase Recombination of Phage l in E. coli

 Int: integrase  IHF: integration host Phage l factor  Xis : Excisonase

E. coli

Lysogen Recombination of Phage l in E. coli  7 bp core region is responsible for specificity

Invitrogen Cloning Using Recombination

ccdB : Encoding toxin

 counter selection marker gene Generated by PCR attL1 attL2 ccdB Entry Clone attP1 attP2 attB1 gene attB2 attR1 ccdB attR2 Donor KanR + Vector +

KanR BP Clonase™ II

 Int, IHF 90-99% correct clones on Kan plates

gene attB1 attB2

ccdB Expression Clone attR1 attR2 Destination AmpR + Vector +

AmpR LR Clonase™ II

 Int, IHF, Xis 90-99% correct clones on Amp plates

Invitrogen