Recombinant DNA

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

E. coli transformation and selection Joining to a vector

Ligation Recombination

Preparation of Insert DNA

Genomic

DNA cDNA PCR

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 system against

Invasion of foreign DNA



Phage fd K



Modified during growth in E.coli K



Restricted in growth in E.coli B

fd

E. coli K

E. coli B fd K

E. coli K

Restriction and Modification

Restriction: Endonuclease

Modification: Methylase

Types of Endonuclease System

System Enzyme Subunits Recognition sites

Cleavage sites

Type I 1

3 (Recognition, Methylation,

Cleavage)

No feature Up to 1 kb away

Type II 2 (Cleavage,

Modification) Symmetrical Recognition site

Type III 1

2 (Recognition and

Modification, Cleavage)

Symmetrical 24-26 bp away

Type IIs 2 (Cleavage,

Modification) Asymmetrical

Up to 20 bp away

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

Serratia marcescencs : species 1

enzyme

Hind III

Haemophilus influenzae : species 3

enzyme

Strain d

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)



Neoschizomer

 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

 Restriction enzymes have different preference for methylated DNA

 e.g. MboI (GATC), DpnI (GA^mTC), Sau3AI (GA^mTC, GATC) EcoRII (CCA/TGG), BstNI (C^mCA/TGG, CCA/TGG)

 Reduced transformation efficiency of methylated DNA to other species: use dam^-, dcm^- strain for DNA preparation

Methylase Recognition Sequence Frequency (if 50% GC)

dam GA^mTC (N⁶) 256 bp

dcm C^mCA/TGG (C⁵) 512 bp

M.EcoKI AA^mCGTGC GCA^mCGTT (N⁶) 8 kb

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

Log₁₀ MW Migration

distance



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 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 g-phosphate from g-

32

P ATP

g³²P-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’

3’ TGGCC/A 5’

5’ A/CCGGT 3’ 5’ C/CCGGG 3’

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’

3’ GGG/CCC 5’ 3’ CTA/TAG5

5’ CCC/ATC 3’ 5’ GAT/GGG 3’

3’ GGG/TAG 5’ 3’ CTA/CCC5’

AgeI

AvaI

+ +

AgeI

+

SmaI EcoRV

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: 4-15^oC: ensure base pairing

 Blunt ends: 18^oC, 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

 Blunt ends DNA containing restriction enzyme site

 Adaptor

 Chemically synthesized DNA with cohesive ends

 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

5’ 5’

3’

3’

R1

R2

PCR

R1 R2

RE

linker

adaptor

Efficiency of Enzyme Digestion

% Cleavage

Enzyme Oligo Sequence Chain length 2 hr 20 hr

BamHI

CGGATCCG CGGGATCCCG CGCGGATCCGCG

8 10 12

10

>90

>90

25

>90

>90

EcoRI

GGAATTCC CGGAATTCCG CCGGAATTCCGG

8 10 12

>90

>90

>90

>90

>90

>90

HindIII

CAAGCTTG CCAAGCTTGG CCCAAGCTTGGG

8 10 12

0 0 10

0 0 75

Sma I

CCCGGG CCCCGGGG CCCCCGGGGG TCCCCCGGGGGA

6 8 10 12

0 0 10

>90

10 10 50

>90

Xba I

CTCTAGAG GCTCTAGAGC TGCTCTAGAGCA CTAGTCTAGACTAG

8 10 12 14

0

>90 75 75

0

>90

>90

>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

Phage l

E. coli

Lysogen

 Int: integrase

 IHF: integration host factor

 Xis : Excisonase

 7 bp core region is responsible for specificity

Invitrogen

Recombination of Phage l in E. coli

90-99% correct clones on Kan plates

Cloning Using Recombination

gene

attB1 attB2

+

attP1 attP2

ccdB

Donor Vector KanR

+

gene

attL1 attL2

Entry Clone ccdB KanR

attR1 attR2

BP Clonase™ II

Generated by PCR

ccdB : Encoding toxin

 counter selection marker

Expression Clone

gene

attB1 attB2

AmpR

attP1 attP2

ccdB

Donor Vector KanR

attR1 attR2

ccdB

Destination Vector

AmpR gene

attL1 attL2

Entry Clone KanR

90-99% correct clones on Amp plates

+ +

LR Clonase™ II

 Int, IHF

 Int, IHF, Xis

Invitrogen