Università degli Studi di Veronaprofs.sci.univr.it/~delledonne/Insegnamenti/Dispense...08/03/09...

UNIVERSITÀ DEGLI STUDI DI VERONA

Corso di Laurea in Biotecnologie Agro-Industriali

Dispense di

BIOTECNOLOGIE GENETICHE

Prof. Massimo Delledonne Tel. 045-8027962; Email: [email protected]

http://www.sci.univr.it/~delledon

08/03/09 Biotecnologie genetiche Massimo Delledonne

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THE GATEWAY® TECHNOLOGY ...................................................................................... 5

Gateway® BP and LR Recombination Reactions ........................................................................... 7 Features of the Gateway® Vectors .................................................................................................. 9

Gateway® Entry Vectors ................................................................................................................. 10

The pDONR Vectors ...................................................................................................................... 10

Designing attB PCR Primers ......................................................................................................... 11 pENTR. Directional TOPO® Cloning ........................................................................................... 14

Gateway® Destination Vectors ........................................................................................................ 23

E. coli Expression System with with Gateway® Technology ....................................................... 24

Gateway vectors for Agrobacterium-mediated plant transformation ............................................ 28

MUTAGENESI ................................................................................................................... 30

Mutagenesi random ......................................................................................................................... 30

Mutagenesi sitodiretta ..................................................................................................................... 32 QuikChange® Stratagene .............................................................................................................. 34

Mutagenesi sitodiretta con ricombinazione in vivo ....................................................................... 36

Phage Display ................................................................................................................................... 37

CDNA ................................................................................................................................ 39

cDNA synthesis and cDNA libraries............................................................................................... 39

cDNA Amplification Using One-Sided, Anchored, PCR (RACE) ............................................... 47

Amplification of regions downstream (3) of known sequence ..................................................... 47

Amplification of regions upstream (5) of known sequence .......................................................... 48

Get the 5'-End: the NEW-RACE (RML-RACE) ........................................................................... 50

SMART™ RACE cDNA Amplification Kit ................................................................................. 52

GENE TAGGING ............................................................................................................... 54

Transposon Tagging ........................................................................................................................ 54

T-DNA tagging ................................................................................................................................. 55

Activation tagging ............................................................................................................................ 55

Thermal Asymmetric Interlaced PCR ........................................................................................... 58

Amplification of insertion mutagenised sites (AIMS) ................................................................... 61

REVERSE GENETICS ...................................................................................................... 63

PCR ................................................................................................................................... 64

Polimerasi alta fedeltà ..................................................................................................................... 65


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Multiplex PCR .................................................................................................................................. 67

Competitive RT-PCR ....................................................................................................................... 68

Real Time PCR ................................................................................................................................. 71 SYBR. Green I Dye Chemistry ...................................................................................................... 73

TaqMan - Real-Time Quantitative PCR ........................................................................................ 76

LUX Real-time or quantitative PCR .............................................................................................. 77

Molecular beacons ......................................................................................................................... 79

Scorpion probes ............................................................................................................................. 85

MOLECULAR MARKERS ................................................................................................. 89 Genetic and Physical maps of the genome..................................................................................... 89

Isoenzymes ........................................................................................................................................ 91

DNA markers based on variable numbers of short sequence repeats. ....................................... 92 Variable Number Tandem repeat (VNTR) .................................................................................... 92

DNA markers based on single nucleotide polymorphisms ........................................................... 98

Advantages and disadvantages of various DNA fingerprinting methods ................................. 112

High-throughput SNP genotyping ................................................................................................ 115 High-throughput assay chemistry ................................................................................................ 115

Detection systems ........................................................................................................................ 118

Future trends in high-throughput genotyping technologies ......................................................... 121

DNA SEQUENCING METHODS ..................................................................................... 122 The Sanger Method (metodo dei dideossi) .................................................................................. 122

Next generation sequencing technology ....................................................................................... 126 Pyrosequencing ............................................................................................................................ 127

454 ................................................................................................................................................ 128

Illumina Genome Analyzer II ...................................................................................................... 134

SOLiD .......................................................................................................................................... 138

Pacific Biosciences ...................................................................................................................... 141

FOR DISCOVERY OF DIFFERENTIALLY EXPRESSED GENES ................................. 147

Differential screening ..................................................................................................................... 148

Production of a Subtracted cDNA Library ................................................................................. 148

PCR-Based Subtractive cDNA Cloning (PCR-Select) ................................................................ 151

Differential Display of mRNA by PCR ........................................................................................ 154

Reverse northern ............................................................................................................................ 157

AFLP-Based Transcript Profiling ................................................................................................ 159


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Painless Gene Expression Profiling: SAGE (Serial Analysis of Gene Expression).................. 165 Supersage ..................................................................................................................................... 167

EST e electronic northern ............................................................................................................. 169 Exhaustive mining of EST libraries for genes differentially expressed....................................... 169

UniGene ....................................................................................................................................... 172

Tissue issues: measurements of gene expression in specific cell types ...................................... 175 Isolation of Cell-Specific RNA and Other Macromolecules by Laser-Capture Microdissection 175

Methods for Transcriptional Profiling ......................................................................................... 177

MICROARRAY ................................................................................................................ 188

Applications of microarray analysis ............................................................................................. 191

Genetic content of microarrays .................................................................................................... 194

Chips of a different flavor ............................................................................................................. 198 cDNA Microarrays ....................................................................................................................... 198

Oligonucleotide Arrays ................................................................................................................ 199

Manufacturing of microarray slides ............................................................................................ 201 Production methods ..................................................................................................................... 201

Requirements of microarray spotting methods and instruments .................................................. 202

Microarray slide types .................................................................................................................. 205

Target nucleic acids ..................................................................................................................... 205

Fluorescent labels in microarray analysis ................................................................................... 211

Preparation of RNA samples for microarray analysis ............................................................... 213

Sample labelling for gene expression analysis ............................................................................. 213 Requirements of labelling methods.............................................................................................. 214

Labelling strategies ...................................................................................................................... 215

Enzymatic labelling methods ....................................................................................................... 216

Microarray hybridization .............................................................................................................. 220 Pre-hybridization .......................................................................................................................... 220

Hybridization ............................................................................................................................... 220

Stringency washes ........................................................................................................................ 221

Fluorescence imaging systems in microarray analysis ............................................................... 221

Design, controls and data analysis of microarray experiments ................................................. 222 Use of controls in microarray experiments .................................................................................. 223

Normalization............................................................................................................................... 224

Visualization and clustering ......................................................................................................... 226

Gene arrays are not just for measuring gene expression ........................................................... 227


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The Gateway® Technology The Gateway® Technology (Invitrogen) is based on the bacteriophage lambda site-specific recombination system

which facilitates the integration of lambda into the E. coli chromosome and the switch between the lytic and lysogenic

pathways. In the Gateway® Technology, the components of the lambda recombination system are modified to improve

the specificity and efficiency of the system. This section provides a brief overview of lambda recombination and the

reactions that constitute the Gateway® Technology.

Recombination Components

Lambda-based recombination involves two major components:

• The DNA recombination sequences (att sites) and

• The proteins that mediate the recombination reaction (i.e. Clonase™ enzyme mix)

These components are discussed below.

Characteristics of the Recombination Reactions

Lambda integration into the E. coli chromosome occurs via intermolecular DNA recombination that is mediated by a

mixture of lambda and E. coli-encoded recombination proteins (i.e. Clonase™ enzyme mix). The hallmarks of lambda

recombination are listed below.

• Recombination occurs between specific attachment (att) sites on the interacting DNA molecules.

• Recombination is conservative (i.e. there is no net gain or loss of nucleotides) and requires no DNA synthesis. The

DNA segments flanking the recombination sites are switched, such that after recombination, the att sites are hybrid

sequences comprised of sequences donated by each parental vector. For example, attL sites are comprised of

sequences from attB and attP sites.

• Strand exchange occurs within a core region that is common to all att sites (see below).

• The recombination can occur between DNAs of any topology (i.e. supercoiled, linear, or relaxed), although efficiency

varies.

att Sites

Lambda recombination occurs between site-specific attachment (att) sites: attB on the E. coli chromosome and attP on

the lambda chromosome. The att sites serve as the binding site for recombination proteins and have been well-

characterized. Upon lambda integration, recombination occurs between attB and attP sites to give rise to attL and attR

sites. The actual crossover occurs between homologous 15 bp core regions on the two sites, but surrounding sequences

are required as they contain the binding sites for the recombination proteins.

Recombination Proteins

Lambda recombination is catalyzed by a mixture of enzymes that bind to specific sequences (att sites), bring together

the target sites, cleave them, and covalently attach the DNA. Recombination occurs following two pairs of strand

exchanges and ligation of the DNAs in a novel form. The recombination proteins involved in the reaction differ

depending upon whether lambda utilizes the lytic or lysogenic pathway (see table below).

The lysogenic pathway is catalyzed by the bacteriophage λ Integrase (Int) and E. coli Integration Host Factor (IHF)

proteins (BP Clonase™ enzyme mix) while the lytic pathway is catalyzed by the bacteriophage λ Int and Excisionase

(Xis) proteins, and the E. coli Integration Host Factor (IHF) protein (LR Clonase™ enzyme mix).

Pathway Reaction Catalyzed by…

Lysogenic attB x attP → attL x attR BP Clonase™ (Int, IHF)

Lytic attL x attR → attB x attP LR Clonase™ (Int, Xis, IHF)

Gateway® Recombination Reactions

The Gateway® Technology uses the lambda recombination system to facilitate transfer of heterologous DNA sequences

(flanked by modified att sites) between vectors (Hartley et al., 2000). Two recombination reactions constitute the basis

of the Gateway® Technology:


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BP Reaction: Facilitates recombination of an attB substrate (attB-PCR product or a linearized attB expression clone) with an attP substrate (donor vector) to create an attL-containing entry clone (see diagram below). This

reaction is catalyzed by BP Clonase™ enzyme mix.

LR Reaction: Facilitates recombination of an attL substrate (entry clone) with an attR substrate (destination vector) to create an attB-containing expression clone (see diagram below). This reaction is catalyzed by LR

Clonase™ enzyme mix.


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Gateway® BP and LR Recombination Reactions The wild-type λ att recombination sites have been modified to improve the efficiency and specificity of the Gateway®

BP and LR recombination reactions. This section describes the modifications and provides examples of the Gateway®

recombination reactions between the attB x attP and attL x attR sites.

Modifications to the att Sites

In the Gateway® System, the wild-type λ att recombination sites have been modified in the following ways to improve

the efficiency and specificity of the Gateway® BP and LR recombination reactions:

Mutations have been made to the core regions of the att sites to eliminate stop codons and to ensure specificity of the recombination reactions to maintain orientation and reading frame.

Mutations have been introduced into the short (5 bp) regions flanking the 15-bp core regions of the attB sites to minimize secondary structure formation in single-stranded forms of attB plasmids (e.g. phagemid ssDNA or

mRNA).

A 43 bp portion of the attR site has been removed to make the in vitro attL x attR reaction irreversible and more efficient.

_________

In addition to the modifications described above, site-specific point mutations have been made to some att sites to

increase recombination efficiency. As a result, sequence variations may exist among the att sites. For example, the

pDONR™201 attP1 sequence varies slightly from the pDONR™221 attP1 sequence. These sequence variations do not

affect the specificity of the recombination reactions or the functionality of the vectors.

Characteristics of the Modified att Sites

The modified att sites have the following characteristics and specificity.

Site Length Found in…

attB 25 bp Expression vector

Expression clone

attP 200 bp Donor vector

attL 100 bp Entry vector

Entry clone

attR 125 bp Destination vector

Specificity:

attB1 sites react only with attP1 sites

attB2 sites react only with attP2 sites

attL1 sites react only with attR1 sites

attL2 sites react only with attR2 sites

Example of an attB x attP Recombination Reaction

The diagram below depicts a BP recombination reaction between an attB-PCR product and the pDONR™221 or

pDONR™/Zeo vector to create an entry clone and a by-product.

Note: If you are performing a BP recombination reaction using a donor vector other than pDONR™221 or

pDONR™/Zeo, note that the sequences of the recombination regions may vary slightly but the mechanism of

recombination remains the same.

Features of the Recombination Region:

Shaded regions correspond to those sequences transferred from the attB-PCR product into the entry clone following recombination. Note that the attL sites are composed of sequences from attB and attP.

Boxed regions correspond to those sequences transferred from pDONR™221 or pDONR™/Zeo into the by-product following recombination.


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Example of an attL x attR Recombination Reaction

The diagram below depicts an LR recombination reaction between a pENTR/DTOPO ® entry clone and the

pcDNA™6.2/V5-DEST destination vector to create an expression clone and a by-product.

Note: If you are performing an LR recombination reaction using different vectors, note that the sequences of the

recombination regions may vary slightly but the mechanism of recombination remains the same.


Shaded regions correspond to those sequences transferred from the pENTR/D-TOPO® entry clone into the expression clone following recombination. Note that the attB sites are composed of sequences from attL and attR

sites.

Boxed regions correspond to those sequences transferred from pcDNA™6.2/V5-DEST into the by-product following recombination.


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Features of the Gateway® Vectors Gateway® Vectors Three different types of Gateway®-adapted vectors are available from Invitrogen:

Gateway® Vector Characteristics

Donor vector (pDONR™) Contains attP sites

Used to clone attB-flanked PCR products and genes

of interest to generate entry clones

Entry vector (pENTR™) Contains attL sites

Used to clone PCR products or restriction fragments

that do not contain att sites to generate entry clones

Destination vector Contains attR sites

Recombines with the entry clone in an LR reaction to

generate an expression clone

Contains elements necessary to express the gene of

interest in the appropriate system (i.e. E. coli,

mammalian, yeast, insect)

Common Features of the Gateway® Vectors

To enable recombinational cloning and efficient selection of entry or expression clones, most Gateway® vectors contain

two att sites flanking a cassette containing:

• The ccdB gene (see below) for negative selection (present in donor, destination, and supercoiled entry vectors)

• Chloramphenicol resistance gene (CmR) for counterselection (present in donor and destination vectors)

After a BP or LR recombination reaction, this cassette is replaced by the gene of interest to generate the entry clone and

expression clone, respectively.

ccdB Gene

The presence of the ccdB gene allows negative selection of the donor and destination (and some entry) vectors in E. coli

following recombination and transformation. The CcdB protein interferes with E. coli DNA gyrase (Bernard and


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Couturier, 1992), thereby inhibiting growth of most E. coli strains (e.g. DH5α™, TOP10). When recombination occurs

(i.e. between a destination vector and an entry clone or between a donor vector and an attB-PCR product), the ccdB

gene is replaced by the gene of interest. Cells that take up unreacted vectors carrying the ccdB gene or by-product

molecules retaining the ccdB gene will fail to grow. This allows high-efficiency recovery of the desired clones.

Propagating Gateway® Vectors

Because of the lethal effects of the CcdB protein, all Gateway® vectors containing the ccdB gene must be propagated

in an E. coli strain that is resistant to CcdB effects. We recommend using the DB3.1™ E. coli strain which contains a

gyrase mutation (gyrA462) that renders it resistant to the CcdB effects (Bernard and Couturier, 1992; Bernard et al.,

1993; Miki et al., 1992). Library Efficiency® DB3.1™ Competent Cells are available from Invitrogen (Catalog no.

11782-018) for transformation. To maintain the integrity of the vector, select for transformants in media containing the

appropriate antibiotic and 15 µg/ml chloramphenicol

Genotype of DB3.1 F- gyrA462 endA1 ∆(sr1-recA) mcrB mrr hsdS20(rB-, mB-) supE44 ara14 galK2 lacY1proA2

rpsL20(Smr) xyl5 ∆leu mtl1

Gateway® Entry Vectors

The pDONR Vectors

The map below shows the elements of pDONR.221 and pDONR./Zeo. The complete sequences of pDONR.221

and pDONR./Zeo are available for downloading at www.invitrogen.com.

Features of pDONR.221 and pDONR./Zeo

pDONR.221 (4762 bp) and pDONR./Zeo (4291 bp) contain the following elements. All features have been functionally

tested.

Feature Benefit

rrnB T1 and T2 transcription terminators Protects the cloned gene from expression by vector-

encoded promoters, thereby reducing possible

toxicity (Orosz et al., 1991)


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M13 Forward (-20) priming site Allows sequencing in the sense orientation

attP1 and attP2 sites Bacteriophage λ-derived DNA recombination

sequences that allow recombinational cloning of the

gene of interest from a Gateway® expression clone

or attB PCR product (Landy, 1989)

ccdB gene Allows negative selection of the plasmid

Chloramphenicol resistance gene (CmR) Allows counterselection of the plasmid

M13 Reverse priming site Allows sequencing in the anti-sense orientation

Kanamycin resistance gene

(pDONR.221 only)

Allows selection of the plasmid in E. coli

EM7 promote

(pDONR./Zeo only)

Allows expression of the Zeocin. Resistance gene in

E. coli.

Zeocin. resistance gene

(pDONR./Zeo only)

Allows selection of the plasmid in E. coli

pUC origin Allows high-copy replication and maintenance in E.

coli

Recombination Region of pDONR.221 and pDONR./Zeo

The recombination region of the expression clone resulting from pDONR.221 × entry clone or pDONR./Zeo × entry

clone is shown below.


• Shaded regions correspond to DNA sequences transferred from the attB substrate into pDONR.221 or pDONR./Zeo

by recombination. Non-shaded regions are derived from the pDONR.221 or pDONR./Zeo vector.

• Bases 651 and 2897 of the pDONR.221 or pDONR./Zeo vector sequence are marked.

Designing attB PCR Primers

To generate PCR products suitable for use as substrates in a Gateway® BP recombination reaction with a donor vector,

you will need to incorporate attB sites into your PCR products. Guidelines are provided below to help you design your

PCR primers.

Designing Your PCR Primers

The design of the PCR primers to amplify your gene of interest is critical for recombinational cloning using Gateway®.

Consider the following when designing your PCR primers:

• Sequences required to facilitate Gateway® cloning

• Sequence required for efficient expression of the native protein (i.e. Shine-Dalgarno or Kozak consensus), if necessary

• Whether or not you wish your PCR product to be fused in frame with an Nor C-terminal fusion tag


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Guidelines to Design the Forward PCR Primer

When designing your forward PCR primer, consider the points below. Refer to the diagram below and Examples 1 and

2 for more help.

• To enable efficient Gateway® cloning, the forward primer MUST contain the

following structure:

1. Four guanine (G) residues at the 5′ end followed by

2. The 25 bp attB1 site followed by

3. At least 18-25 bp of template- or gene-specific sequences

Note: If you plan to express native protein in E. coli or mammalian cells, you may want to include a Shine-

Dalgarno or Kozak consensus sequence, respectively, in your PCR primer (see Example 1).

• The attB1 site ends with a thymidine (T). If you wish to fuse your PCR product in frame with an N-terminal tag, the

primer must include two additional nucleotides to maintain the proper reading frame with the attB1 region (see

diagram below and Example 2). These two nucleotides cannot be AA, AG, or GA, because these additions will

create a translation termination codon.

Example 1: Forward Primer Design for Native Expression

In this example, we design the following forward attB PCR primer to allow expression of native protein of interest. The

attB1 site is indicated in bold and the ATG initiation codon for the protein of interest is underlined. Inclusion of the

Shine-Dalgarno and Kozak consensus sequence allows protein expression in both

E. coli and mammalian cells.

Note: The ATG initiation codon in this example is in frame with the attB1 sequence, so the PCR product can also be

expressed from an N-terminal fusion destination vector.

Example 2: Forward Primer Design for N-terminal Fusions

In this example, we design the following forward attB PCR primer to allow expression of an N-terminal fusion protein

of interest. The attB1 site is indicated in bold. Remember that the gene-specific nucleotides need to be in frame with the

attB1 sequence and that no stop codons should be introduced.

Tip: Keep the -AAA-AAA- triplets in the attR1 site in frame with the translation reading frame of the fusion protein.

Guidelines to Design the Reverse PCR Primer

When designing your reverse PCR primer, consider the points below. Refer to the diagram below and Examples 1 and

2, next page for more help.

• To enable efficient Gateway® cloning, the reverse primer MUST contain the following structure:

1. Four guanine (G) residues at the 5′ end followed by

2. The 25 bp attB2 site followed by

3. 18-25 bp of template- or gene-specific sequences

• If you wish to fuse your PCR product in frame with a C-terminal tag:


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1. The primer must include one additional nucleotide to maintain the proper reading frame with the attB2 region

(see diagram below and Example 2, next page)

2. Any in-frame stop codons between the attB2 site and your gene of interest must be removed

• If you do not wish to fuse your PCR product in frame with a C-terminal tag, your gene of interest or the primer must

include a stop codon (see Example 1, next page)

Example 1: Reverse Primer Design

In this example, we design the following reverse attB PCR primer to allow expression of a protein of interest with no C-

terminal fusion tag. The attB2 site is indicated in bold and the stop codon for the protein of interest is underlined.

Remember that the gene-specific nucleotides need to be in frame with the stop codon.

Example 2: Reverse Primer Design for C-terminal Fusions

In this example, we design the following reverse attB PCR primer to allow expression of a C-terminal fusion protein of

interest. The attB2 site is indicated in bold. Remember that the gene-specific nucleotides need to be in frame with the

attB2 sequence and that stop codons should be removed.

Tip: Keep the -TTT-GTA (TAC-AAA on the complementary strand) triplets in the attR2 site in frame with the

translation reading frame of the fusion protein.

Producing PCR Products

Standard PCR conditions can be used to prepare attB-PCR products. Follow the manufacturer‘s instructions for the

DNA polymerase you are using, and use the cycling parameters suitable for your primers and template.


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pENTR. Directional TOPO® Cloning

Five-minute, directional TOPO® Cloning of blunt-end PCR products into an entry vector for the

Gateway® System

The pENTR™ Directional TOPO® Cloning Kits utilize a highly efficient, 5-minute cloning strategy ("TOPO®

Cloning") to directionally clone a blunt-end PCR product into a vector for entry into the Gateway® System or the

MultiSite Gateway® System available from Invitrogen. Blunt-end PCR products clone directionally at greater than 90%

efficiency, with no ligase, post-PCR procedures, or restriction enzymes required.

A choice of pENTR™ Directional TOPO® vectors is available for optimal expression of your PCR product after

recombination with the Gateway® destination vector of interest (see table below).

Vector Benefit

pENTR™/D-TOPO® For efficient expression of your gene of interest after

recombination with a Gateway® destination vector

pENTR™/SD/D-TOPO® Contains a T7 gene 10 translational enhancer and a

ribosome binding site (RBS) for optimal expression of

native protein after recombination with a prokaryotic

Gateway® destination vector

Note: Also suitable for efficient expression of your gene

of

interest in other hosts after recombination with a

Gateway®

destination vector (e.g mammalian, insect, yeast)

pENTR™/TEV/DTOPO® Contains a Tobacco Etch Virus (TEV) recognition site

for efficient TEV protease-dependent cleavage of an

N-terminal tag from your recombinant protein after

recombination and expression from a Gateway®

destination vector

Features of the pENTR. TOPO® Vectors

The pENTR™/D-TOPO®, pENTR™/SD/D-TOPO®, and pENTR™/TEV/D-TOPO® vectors are designed to facilitate

rapid, directional TOPO® Cloning of blunt-end PCR products for entry into the Gateway® System. Features of the

vectors include:

attL1 and attL2 sites for site-specific recombination of the entry clone with a Gateway® destination vector

T7 gene 10 translation enhancer and ribosome binding site for efficient translation of the PCR product in prokaryotes (pENTR™/SD/D-TOPO® only)

TEV recognition site for TEV protease-dependent cleavage of an N-terminal tag from your recombinant protein (pENTR™/TEV/D-TOPO® only)

Directional TOPO® Cloning site for rapid and efficient directional cloning of blunt-end PCR products (see page 3 for more information)

rrnB transcription termination sequences to prevent basal expression of the PCR product of interest in E. coli

Kanamycin resistance gene for selection in E. coli

pUC origin for high-copy replication and maintenance of the plasmid in E. coli

How Topoisomerase I Works Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites (CCCTT) and cleaves the phosphodiester backbone in one strand. The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phosphotyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). Therefore, DNA topoisomerase I functions as both a restriction enzyme and a ligase. Its biological role is to cleave and rejoin DNA during replication. To harness the religating activity of topoisomerase, over 30 vectors are provided linearized with topoisomerase I covalently bound to each 3' phosphate. This enables fast ligation of DNA sequences with compatible ends. After only 5 minutes at room temperature, the ligation is complete and ready for transformation into E. coli.

Directional TOPO® Cloning


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Directional joining of double-strand DNA using TOPO®-charged oligonucleotides occurs by adding a 3′ single-

stranded end (overhang) to the incoming DNA (Cheng and Shuman, 2000). This single-stranded overhang is identical to

the 5′ end of the TOPO®-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide

overhang sequence to the TOPO®-charged DNA and adapting it to a ‗whole vector‘ format. In this system, PCR

products are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning

vector (GTGG) invades the 5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the

correct orientation. Inserts can be cloned in the correct orientation with efficiencies equal to or greater than 90%.

The 5′ TOPO® recognition site in pENTR™/TEV/D-TOPO® is encoded by the sequence TCCTT rather than CCCTT.

This is because the 5′ TOPO® recognition site directly follows the TEV recognition site, and studies have shown that

TEV protease does not cleave efficiently if the first amino acid following the TEV recognition sequence is proline

(Kapust et al., 2002) as would be the case if the 5′ TOPO® recognition site was encoded by CCCTT. By changing the

sequence of the 5′ TOPO® recognition site to TCCTT, the first amino acid following the TEV recognition site is now

serine. This change does not affect TOPO® Cloning efficiency and allows efficient TEV cleavage.

Designing Your PCR Primers

The design of the PCR primers to amplify your gene of interest is critical for expression. Depending on the pENTR™

TOPO® vector you are using, consider the following when designing your PCR primers.

• Sequences required to facilitate directional cloning

• Sequences required for proper translation initiation of your PCR product

• Whether or not you wish your PCR product to be fused in frame with an Nor C-terminal tag after recombination of

your entry clone with a Gateway® destination vector

Guidelines to Design the Forward PCR Primer

When designing your forward PCR primer, consider the following points below.

To enable directional cloning, the forward PCR primer must contain the sequence, CACC, at the 5′ end of the primer. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in each pENTR™ TOPO®

vector.

If you plan to express your PCR product in mammalian cells as a native or C-terminal fusion-tagged protein (following recombination of the entry clone with a Gateway® destination vector), your sequence of interest should

include a Kozak translation initiation sequence with an ATG initiation codon for proper initiation of translation

(Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of a Kozak consensus sequence is (G/A)NNATGG. Other

sequences are possible, but the G or A at position –3 and the G at position +4 are the most critical for function

(shown in bold). The ATG initiation codon is underlined. Note: If your sequence of interest does not contain an

initiation codon within the context of a Kozak sequence, design the forward PCR primer to contain a Kozak

sequence at the 5′ end of the primer (see Example on the next page).

If you plan to express your PCR product in mammalian cells as an N-terminal fusion-tagged protein (following recombination of the entry clone with a Gateway® destination vector), your sequence of interest does not need to

contain a Kozak translation initiation sequence. A Kozak sequence is provided by the appropriate destination

vector. Note: In this case, internal initiation may occur if your PCR product contains an endogenous Kozak

sequence.

If you plan to express your PCR product in prokaryotic cells without an N-terminal fusion tag (following recombination of the entry clone with a Gateway® destination vector), you should TOPO® Clone your PCR

product into pENTR™/SD/D-TOPO®. pENTR™/SD/D-TOPO® contains a T7 gene 10 translational enhancer and


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a ribosome binding site (RBS) to enable efficient translation of the PCR product in E. coli. To ensure optimal

spacing for proper translation, design your forward PCR primer so that the ATG initiation codon of your

PCR product directly follows the CACC necessary for directional cloning (see Example on the next page).

Example of Forward Primer Design

Below is the DNA sequence of the N-terminus of a theoretical protein and the proposed sequence for your forward PCR

primer. The ATG initiation codon is underlined.

DNA sequence: 5′-ATG GGA TCT GAT AAA

Proposed Forward PCR primer: 5′-C ACC ATG GGA TCT GAT AAA

If you design the forward PCR primer as noted above, then:

The ATG initiation codon falls within the context of a Kozak sequence (see boxed sequence), allowing proper translation initiation of the PCR product in mammalian cells.

The ATG initiation codon is properly spaced from the RBS (in pENTR™/SD/D-TOPO® only), allowing proper translation of the PCR product in prokaryotic cells. The first three base pairs of the PCR product following the 5′

CACC overhang will constitute a functional codon.

Guidelines to Design the Reverse Primer

When designing your reverse PCR primer, consider the following points below.

To ensure that your PCR product clones directionally with high efficiency, the reverse PCR primer MUST NOT be complementary to the overhang sequence GTGG at the 5′ end. A one base pair mismatch can

reduce the directional cloning efficiency from 90% to 50%, increasing the likelihood of your ORF cloning in

the opposite orientation (see Example #1 on the next page). We have not observed evidence of PCR products

cloning in the opposite orientation from a two base pair mismatch.

If you wish to fuse your PCR product in frame with a C-terminal tag (following recombination of the entry clone with a Gateway® destination vector), then design the reverse PCR primer to remove the native stop codon in the

gene of interest (see Example #2 on the next page).

If you do not wish to fuse your PCR product in frame with a C-terminal tag (following recombination of the entry clone with a Gateway® destination vector), then include the native sequence containing the stop codon in the

reverse primer or make sure the stop codon is upstream from

Example #1 of Reverse Primer Design

Below is the sequence of the C-terminus of a theoretical protein. You want to fuse the protein in frame with a C-

terminal tag (following recombination of the entry clone with a Gateway® destination vector). The stop codon is

underlined.

DNA sequence: AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′

One solution is to design the reverse PCR primer to start with the codon just upstream of the stop codon, but the last

two codons contain GTGG (underlined below), which is identical to the 4 bp overhang sequence. As a result, the

reverse primer will be complementary to the 4 bp overhang sequence, increasing the probability that the PCR product

will clone in the opposite orientation. You want to avoid this situation.

DNA sequence: AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′

Proposed Reverse PCR primer sequence: TG AGC TGC TGC CAC AAA-5′

Another solution is to design the reverse primer so that it hybridizes just downstream of the stop codon, but still

includes the C-terminus of the ORF. Note that you will need to replace the stop codon with a codon for an innocuous

amino acid such as glycine, alanine, or lysine.

Example #2 of Reverse Primer Design

Below is the sequence for the C-terminus of a theoretical protein. The stop codon is underlined.

…GCG GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG-3′

To fuse the ORF in frame with a C-terminal tag (supplied by the destination vector after recombination), remove the stop codon by starting with nucleotides homologous to the last codon (TGC) and continue upstream. The

reverse primer will be: 5′-TGC AGT CGT CGA GTG CTC CGA CTT-3′


17

This will amplify the C-terminus without the stop codon and allow you to join the ORF in frame with a C-

terminal tag.

If you don‘t want to join the ORF in frame with a C-terminal tag, simply design the reverse primer to include the stop codon.

5′-CTA TGC AGT CGT CGA GTG CTC CGA CTT-3′

Important: Remember that the pENTR™ TOPO® vectors accept blunt-end PCR products. • Do not add 5´ phosphates

to your primers for PCR. This will prevent ligation into the pENTR™ TOPO® vectors. • We recommend that you gel-

purify your oligonucleotides, especially if they are long (> 30 nucleotides).

TOPO® Cloning Site for pENTR./D-TOPO®

Use the diagram below to help you design suitable PCR primers to clone your PCR product into pENTR™/D-TOPO®.

Restriction sites are labeled to indicate the actual cleavage site. The shaded region corresponds to the DNA sequences

that will be transferred from the entry clone into the destination vector following LR recombination. The sequence of

pENTR™/D-TOPO® is available for downloading from Invitrogen Web site (www.invitrogen.com)

TOPO® Cloning Site for pENTR./SD/DTOPO®

Use the diagram below to help you design suitable PCR primers to clone your PCR product into pENTR™/SD/D-

TOPO®. Restriction sites are labeled to indicate the actual cleavage site. The shaded region corresponds to the DNA

sequences that will be transferred from the entry clone into the destination vector following LR recombination. The

sequence of pENTR™/D-TOPO® is available for downloading from Invitrogen Web site (www.invitrogen.com)

TOPO® Cloning Site for pENTR./TEV/D-TOPO® Use the diagram below to help you design suitable PCR primers to clone your PCR product into pENTR™/TEV/D-

TOPO®. Restriction sites are labeled to indicate the actual cleavage site. The shaded region corresponds to the DNA

sequences that will be transferred from the entry clone into the destination vector following LR recombination.


18

Note: The sequence of the 5′ TOPO® recognition site has been been changed from CCCTT to TCCTT, resulting in an

amino acid substitution of serine for proline. This amino acid change increases the efficiency of TEV protease

cleavage (Kapust et al., 2002), but does not affect the efficiency of TOPO® Cloning.

Producing Blunt-End PCR Products

Once you have decided on a PCR strategy and have synthesized the primers, produce your blunt-end PCR product using

any thermostable, proofreading polymerase. Follow the guidelines below to produce your blunt-end PCR product.

Set up a 25 µl or 50 µl PCR reaction using the guidelines below.

Follow the instructions and recommendations provided by the manufacturer of your thermostable, proofreading polymerase to produce blunt-end PCR products.

Use the cycling parameters suitable for your primers and template. Make sure to optimize PCR conditions to produce a single, discrete PCR product.

Use a 7 to 30 minute final extension to ensure that all PCR products are completely extended.

After cycling, place the tube on ice or store at –20ºC for up to 2 weeks.

Setting Up the TOPO® Cloning Reaction

Once you have produced the desired blunt-end PCR product, you are ready to TOPO® Clone it into the pENTR™

TOPO® vector and transform the recombinant vector into One Shot® TOP10 E. coli. You should have everything you

need set up and ready to use to ensure that you obtain the best possible results.

Amount of PCR Product to Use in the TOPO® Cloning Reaction

When performing directional TOPO® Cloning, we have found that the molar ratio of PCR product:TOPO® vector used

in the reaction is critical to its success. To obtain the highest TOPO® Cloning efficiency, use a 0.5:1 to 2:1 molar

ratio of PCR product:TOPO® vector (see figure below). Note that the TOPO® Cloning efficiency decreases

significantly if the ratio of PCR product: TOPO® vector is 5:1 (see figure below). These results are

generally obtained if too little PCR product is used (i.e. PCR product is too dilute) or if too much PCR product is used

in the TOPO® Cloning reaction. If you have quantitated the yield of your PCR product, you may need to adjust the

concentration of your PCR product before proceeding to TOPO® Cloning.

Tip: For pENTR™ TOPO® vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a

TOPO® Cloning reaction generally results in a suitable number of colonies.


19

Using Salt Solution in the TOPO® Cloning Reaction

You will perform TOPO® Cloning in a reaction buffer containing salt (i.e. using the stock salt solution provided in the

kit). Note that the amount of salt added to the TOPO® Cloning reaction varies depending on whether you plan to

transform chemically competent cells or electrocompetent cells.

If you are transforming chemically competent E. coli, use the stock Salt Solution as supplied and set up the TOPO® Cloning reaction as directed below.

If you are transforming electrocompetent E. coli, the amount of salt in the TOPO® Cloning reaction must be reduced to 50 mM NaCl, 2.5 mM MgCl2 to prevent arcing during electroporation. Dilute the stock Salt Solution 4-

fold with water to prepare a 300 mM NaCl, 15 mM MgCl2 Dilute Salt Solution. Use the Dilute Salt Solution to set

up the TOPO® Cloning reaction as directed below.

Performing the TOPO® Cloning Reaction

Use the procedure below to perform the TOPO® Cloning reaction. Set up the TOPO® Cloning reaction depending on

whether you plan to transform chemically competent E. coli or electrocompetent E. coli. Reminder: For optimal

results, be sure to use a 0.5:1 to 2:1 molar ratio of PCR product:TOPO® vector in your TOPO® Cloning reaction.

Note: The blue color of the TOPO® vector solution is normal and is used to visualize the solution.

Reagents* Chemically Competent E. coli Electrocompetent E. coli

Fresh PCR product

Salt Solution

Dilute Salt Solution (1:4)

Sterile Water

TOPO® vector

5 to 4 µl

0. 1 µl

--

add to a final volume of 5 µl

1 µl

0.5 to 4 µl

--

1 µl

add to a final volume of 5 µl

1 µl

Final volume 6 µl 6 µl

*Store all reagents at –20°C when finished. Salt solution and water can be stored at room temperature or +4°C.

1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C). Note: For most applications, 5 minutes will yield a sufficient number of colonies for analysis. Depending on your needs, the

length of the TOPO® Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products,

30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO® Cloning a pool of PCR products,

increasing the reaction time may yield more colonies.

2. Place the reaction on ice and proceed to Transforming One Shot® TOP10 Competent Cells, next page. Note: You may store the TOPO® Cloning reaction at –20°C overnight.

One Shot® TOP10 Chemical Transformation Protocol

1. Add 2 µl of the TOPO® Cloning reaction from Performing the TOPO® Cloning Reaction, Step 2, page 12 into a vial of One Shot® TOP10 Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 5 to 30 minutes. Note: Longer incubations on ice seem to have a minimal effect on transformation

3. efficiency. The length of the incubation is at the user‘s discretion. 4. Heat-shock the cells for 30 seconds at 42°C without shaking. 5. Immediately transfer the tubes to ice. 6. Add 250 µl of room temperature S.O.C. Medium. 7. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour. 8. Spread 50-200 µl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. We

recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.

9. An efficient TOPO® Cloning reaction may produce several hundred colonies.

Transformation by Electroporation

1. Add 2 µl of the TOPO® Cloning reaction from Performing the TOPO® Cloning Reaction into a sterile microcentrifuge tube containing 50 µl of electrocompetent E. coli and mix gently. Do not mix by pipetting up and

down. Avoid formation of bubbles. Transfer the cells to a0.1 cm cuvette.

2. Electroporate your samples using your own protocol and your electroporator. 3. Immediately add 250 µl of room temperature S.O.C. Medium. 4. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at least 1 hour at 37°C to allow expression

of the kanamycin resistance gene.


20

5. Spread 20-100 µl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. To ensure even spreading of small volumes, add 20 µl of S.O.C. Medium. We recommend that you plate two different

volumes to ensure that at least one plate will have well-spaced colonies.

6. An efficient TOPO® Cloning reaction may produce several hundred colonies. 7. Pick 5-10 colonies for analysis

Map and Features of pENTR./D-TOPO®

pENTR™/D-TOPO® (2580 bp) contains the following elements. All features have been functionally tested.

Feature Benefit

rrnB T1 and T2 transcription termination

sequences

Reduces potential toxicity in E. coli by

preventing basal expression of the PCR

product.

M13 forward (-20) priming site Allows sequencing of the insert.

attL1 and attL2 sites Bacteriophage λ-derived recombination

sequences that allow recombinational

cloning of a gene of interest in the entry

construct with a Gateway® destination

vector (Landy, 1989).

TOPO® Cloning site (directional) Allows rapid, directional cloning of your

PCR product.

M13 reverse priming site Allows sequencing of the insert.

Kanamycin resistance gene Allows selection of the plasmid in E. coli.

pUC origin of replication (ori) Allows high-copy replication and

maintenance in E. coli.


21

Map and Features of pENTR./SD/D-TOPO®

pENTR™/SD/D-TOPO® (2601 bp) contains the following elements. All features have been functionally tested

Feature Benefit


sequences



product.


T7 gene 10 translational enhancer

Sequence from bacteriophage T7 gene 10

that optimizes translation initiation (Olins et

al., 1988).

Ribosome binding site (RBS) Optimally spaced from the TOPO® Cloning

site for efficient translation of the PCR

product.







PCR product.






22

Map and Features of pENTR./TEV/D-TOPO®

pENTR™/TEV/D-TOPO® (2610 bp) contains the following elements. All features have been functionally tested.

Feature Benefit


sequences



product.







TEV recognition site

Allows removal of the N-terminal tag from

your recombinant protein using AcTEV™

protease (Carrington and Dougherty, 1988;

Dougherty et al., 1988)


PCR product.






23

Gateway®

Destination Vectors You may easily convert any vector of choice to a Gateway® destination vector by ligating a blunt-ended cassette

containing attR sites flanking the ccdB gene and the chloramphenicol resistance gene into the multiple cloning site

(MCS) of the vector. The Gateway® Vector Conversion System is available from Invitrogen to facilitate conversion of

your vector into a destination vector (see below) for expression of native, N-, or C-terminally-tagged proteins.

Most entry vectors contain the kanamycin resistance gene for selection. For maximal compatibility within the

Gateway® Technology, we recommend that your vector not contain a kanamycin resistance marker. If this is

unavoidable, you will need to perform the LR recombination reaction with an entry clone that carries a

selection marker other than the kanamycin resistance gene.

You may use pDONR™/Zeo and your attB-flanked gene of interest in a BP recombination reaction to generate an entry

clone that confers Zeocin™ resistance.

Gateway® Vector Conversion System

The Gateway® Vector Conversion System includes three conversion cassettes:

• Reading Frame (Rf) Cassette A (RfA)

• Reading Frame (Rf) Cassette B (RfB)

• Reading Frame (Rf) Cassette C.1 (RfC.1)

Each reading frame cassette contains the chloramphenicol resistance gene (CmR) and the ccdB gene flanked by attR1

and attR2 sites (see below). Each reading frame cassette also differs by one nucleotide to allow generation of attR sites

in all three reading frames.

Each reading frame cassette contains a unique restriction site to allow you to distinguish between them (see table

below).

Cassette Restriction Site Location (bp) RfA Mlu I 898

RfB Bgl II 899

RfC.1 Xba I 899

Ligating the Reading Frame Cassette to Your Vector

Each reading frame cassette is supplied as a blunt-ended DNA fragment that can be ligated into any blunt-ended

restriction site. It is possible to linearize your vector using a restriction enzyme that generates 5′ overhangs, however,

the ends of the vector must first be made blunt (using a Klenow fill-in reaction) before the

blunt-ended reading frame cassette may be ligated into the vector.

Experimental Outline

To convert your vector to a destination vector, you will:

1. Choose an appropriate reading frame cassette depending on your needs.

2. Linearize the vector with a restriction enzyme of choice. If you use a restriction enzyme that generates an overhang,

you will need to blunt the ends.

3. Remove the 5′ phosphates using calf intestinal alkaline phosphatase.

4. Ligate the reading frame cassette into your vector using T4 DNA ligase.

5. Transform the ligation reaction into DB3.1™ E. coli and select for transformants.

6. Analyze transformants.


24

E. coli Expression System with with Gateway® Technology

Gateway®-adapted destination vectors for cloning and high-level expression of native or tagged recombinant

proteins in E. coli

The E. coli Expression System with Gateway® Technology contains a series of Gateway®-adapted destination vectors

designed to facilitate high-level, inducible expression of recombinant proteins in E. coli using the pET system.

Depending on the vector chosen, the pDEST™ vectors allow production of native, N-terminal, or C-terminal-tagged

recombinant proteins (see table below).

Vector Fusion Peptide Fusion Tag

pDEST™14 --- ---

pDEST™15 N-terminal Glutathione S-transferase (GST)

pDEST™17 N-terminal 6xHis

pDEST™24 C-terminal Glutathione S-transferase (GST)

The pET Expression System

The pET system was originally developed by Studier and colleagues and takes advantage of the high activity and

specificity of the bacteriophage T7 RNA polymerase to allow regulated expression of heterologous genes in E. coli

from the T7 promoter (Rosenberg et al., 1987; Studier and Moffatt, 1986; Studier et al., 1990).

The Basis of T7-Regulated Expression

The pET expression system uses elements from bacteriophage T7 to control expression of heterologous genes in E. coli.

In the pDEST™14, pDEST™15, pDEST™17, and pDEST™24 vectors, expression of the gene of interest is controlled

by a strong bacteriophage T7 promoter. In bacteriophage T7, the T7 promoter drives expression of gene 10 (10). T7

RNA polymerase specifically recognizes this promoter. To express the gene of interest, it is necessary to deliver T7

RNA polymerase to the cells by inducing expression of the polymerase or infecting the cell with phage expressing the

polymerase. In the E. coli Expression System with

Gateway® Technology, T7 RNA polymerase is supplied by the BL21-AI™ host E. coli strain in a regulated manner.

The BL21-AI. E. coli Strain

The BL21-AI™ E. coli strain is intended for use as a host for expression of T7 RNA polymerase-regulated genes. The

BL21-AI™ strain is derived from the BL21 strain (Grodberg and Dunn, 1988; Studier and Moffatt,

1986) and contains a chromosomal insertion of the gene encoding T7 RNA polymerase (T7 RNAP) into the araB locus

of the araBAD operon, placing regulation of the T7 RNAP gene under the control of the araBAD promoter. The araB

gene is deleted in this strain.

Regulating Expression of T7 RNA Polymerase

Because the T7 RNAP gene is inserted into the araB locus of the araBAD operon, expression of T7 RNA polymerase

can be regulated by the sugars, L-arabinose and glucose.

To induce expression from the araBAD promoter, use L-arabinose (Lee, 1980; Lee et al., 1987). To modulate expression, simply vary the concentration of L-arabinose added.

To repress basal expression from the araBAD promoter, use glucose.

Note: In the absence of glucose, basal expression from the araBAD promoter is generally low (Lee, 1980; Lee et al.,

1987). Adding glucose further represses expression from the araBAD promoter by reducing the levels of 3´, 5´-cyclic

AMP (Miyada et al., 1984).

Regulation by L-Arabinose

The araBAD promoter (PBAD) used to control expression of T7 RNA polymerase in BL21-AI™ is both positively and

negatively regulated by the product of the araC gene (Ogden et al., 1980; Schleif, 1992). AraC is a transcriptional

regulator that forms a complex with L-arabinose. In the absence of L-arabinose the AraC dimer contacts the O2 and I1

half sites of the araBAD operon, forming a 210 bp DNA loop (see figure below). For maximum transcriptional

activation two events are required.

L-Arabinose binds to AraC and causes the protein to release the O2 site and bind the I2 site which is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin.

The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2.


25

Glucose Repression

Basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering

cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is

decreased.

Features of the Vectors

pDEST™14, pDEST™15, pDEST™17, and pDEST™24 contain the following elements:

T7 promoter for high-level, T7 RNA polymerase regulated expression of the gene of interest in E. coli (Studier and Moffatt, 1986; Studier et al., 1990)

N- or C-terminal fusion tags for detection and purification of recombinant fusion proteins (choice of tag depends on the particular vector; see above)

Two recombination sites, attR1 and attR2, downstream of the T7 promoter for recombinational cloning of the gene of interest from an entry clone

Chloramphenicol resistance gene (CmR) located between the two attR sites for counterselection

The ccdB gene located between the attR sites for negative selection

Ampicillin resistance gene for selection in E. coli

pBR322 origin for low-copy replication and maintenance of the plasmid in E. coli


26


27

Features of the Vectors

pDEST™14 (6422 bp), pDEST™15 (7013 bp), pDEST™17 (6354 bp), and pDEST™24 (6961 bp) contain the following

elements. All features have been functionally tested.

Feature Benefit

T7 promoter Permits high-level, IPTG-inducible expression of your

recombinant protein in E. coli strains expressing the T7

RNA polymerase.

Ribosome binding site (i.e. Shine-Dalgarno sequence)

(in pDEST™15 and pDEST™17 only)

Optimally spaced from the initiation ATG in the N-terminal

tag for efficient translation of the PCR product.

N-terminal glutathione S-transferase (GST) tag

(in pDEST™15 only)

Allows affinity purification of recombinant fusion protein

using glutathione agarose

N-terminal 6xHis tag

(in pDEST™17 only)

Permits affinity purification of recombinant fusion protein

using a metal-chelating resin such as ProBond™ or Ni-NTA

attR1 and attR2 sites Bacteriophage -derived DNA recombination sequences that

permit recombinational cloning of the gene of interest from a

Gateway® entry clone (Landy, 1989)

Chloramphenicol resistance gene (CmR) Allows counterselection of the plasmid.

ccdB gene Permits negative selection of the plasmid.

C-terminal glutathione S-transferase

(GST) tag (in pDEST™24 only)

Allows affinity purification of recombinant fusion protein

using glutathione agarose

T7 transcription termination region Sequence from bacteriophage T7 which permits efficient

transcription termination.

bla promoter Allows expression of the ampicillin resistance gene.

Ampicillin resistance gene (-lactamase) Allows selection of the plasmid in E. coli.

pBR322 origin of replication (ori) Permits replication and maintenance in E. coli.

ROP ORF Interacts with the pBR322 origin to facilitate low-copy

replication in E. coli.

Points to Consider Before Recombining into pDEST.15 and pDEST.17

pDEST™15 and pDEST™17 are N-terminal fusion vectors and contain an ATG initiation codon upstream of the GST

and 6xHis tags, respectively. In each vector, a Shine-Dalgarno RBS is included upstream of the initiation ATG to

ensure optimal spacing for proper translation initiation in E. coli. Your gene of interest in the entry clone must:

Be in frame with the N-terminal tag after recombination.

Contain a stop codon.

Points to Consider Before Recombining into pDEST.24

pDEST™24 is a C-terminal fusion vector. Your gene of interest in the entry clone must:

Contain an ATG initiation codon and a Shine-Dalgarno RBS with optimal spacing for proper translation initiation in E. coli.

Note: If you clone your gene of interest into an entry vector that supplies a Shine-Dalgarno RBS (e.g.

pENTR/SD/D-TOPO® or pENTR™11), then your gene of interest need only include an ATG initiation codon.

Be in frame with the C-terminal GST tag after recombination.

NOT contain a stop codon.

First transform in E. coli DH5α, then transfer the plasmid into BL21 and express the protein


28

Gateway vectors for Agrobacterium-mediated plant transformation (from Trends in Plant Science 2002, 7: 193-195)

Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the

genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can

replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming

and laborious. Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the

cloning of sequences into large acceptor plasmids.

The backbone of all described GATEWAY™-compatible vectors is the plasmid pPZP200 [1]. This plasmid is relatively

small (6.7 kb), contains an origin for replication in E. coli (ColE1) and in Agrobacterium (pVS1), has a pBR322 bom

site for mobilization from E. coli to Agrobacterium, and possesses a streptomycin and/or spectinomycin resistance gene

for plasmid selection. The T-DNA border regions are derived from the nopaline plasmid pTiT37 and contain the 25 bp

repeat and outer border regions but no internal T-DNA sequences; therefore, natural T-DNA sequences are not

transferred into transformed plants.


29

Destination vectors

All types of T-DNA destination vectors are available with three plant selectable marker genes: the neomycin

phosphotransferase II (nptII), the hygromycin phosphotransferase ( hpt), and the bialaphos acetyltransferase (bar)

genes, which confer resistance against kanamycin, hygromycin and glufosinate ammonium, respectively. All three

selectable markers are under transcriptional regulation of the nopaline synthase ( nos) promoter and nos terminator [2]

and are placed at the left side of the T-DNA.The GATEWAY recombination site for introduction of the fragment of

interest was placed at the right side of the T-DNA. For construction of all vectors in this collection we used the reading

frame A (rfA) of the GATEWAY vector conversion system (Invitrogen).For overexpression of a DNA sequence, the

GATEWAY site was placed between the promoter and the terminator of the cauliflower mosaic virus (CaMV) 35S

transcript [3] because that promoter is highly active in most plant cells of transgenic plants. Downstream of the 35S

promoter part, the tobacco mosaic virus (TMV) W leader ensures efficient translation of the inserted coding sequences

[4]. When both overexpression and cosuppression of a cloned sequence are desired, the p*2GW7 and p*2WG7

destination vectors should be used. In these vectors, the inserted sequences are transcribed to the outside of the T-DNA.

Single T-DNA inserts will then usually result in overexpression of the transgene whereas specially inverted T-DNA

repeats about the right border will trigger post-transcriptional silencing of the transgene and concomitant cosuppression

of homologous endogenes. By contrast, when only overexpression is desired, p*7WG2 is the vector of choice. All

overexpression vectors can also be used for antisense expression of a particular sequence.

A T-DNA destination overexpression or antisense vector (p*7WGD2) with an additional screenable marker was made

essentially for the transformation of tobacco Bright Yellow 2 (BY2), because early visualization of transgenic calli is

important. The visible marker cassette contained the rolD promoter fused to the coding sequences of the enhanced

green-fluorescent protein (GFP) linked to the endoplasmic reticulum-targeting signal (EgfpER) and 35S terminator.

For cosuppression of plant endogenes, two series of destination vectors p*7GWIWG2(I) and p*7GWIWG2(II) were

designed in a way similar to that of the GATEWAY cassette described by S. Varsha Wesley et al. [5]. The intron

cloned between the inverted GATEWAY fragments was selected from an Arabidopsis database (ac007123.em_pl) on

the basis of ideal features for efficient splicing (A + T-rich with a branch site close to the consensus). In the

p*7GWIWG2 (II) series, the chloramphenicol resistance (CmR) marker was removed from the GATEWAY cassette

and placed inside the intron. In plants, these T-DNA destination expression vector constructs will produce double-

stranded RNA (hairpin RNA) from the inserted sequence of interest, triggering post-transcriptional gene silencing in an

efficient way.The p*GWFS7 vectors were constructed for promoter analysis. In these vectors, a frame fusion between

the regions coding for EgfpER and b-glucuronidase (gus) were cloned downstream of the GATEWAY™ cassette. For

the localization of particular proteins, another series of T-DNA destination vectors was designed to allow the N-, C-, or

N- and C-termini of the protein of interest to be fused to the GFP protein.Finally, the T-DNA destination vector p*WG

allows the rapid cloning of any gene or sequence to be transformed into plants. As a test, the gus-coding sequence was

successfully recombined in all sets of T-DNA destination vectors. Where applicable, the expected expression was

observed in transformed hairy roots or BY2 cells (for an update see http://www.plantgenetics.rug.ac.be/gateway/ ).

1. Hajdukiewicz P., et al. "The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation",

Plant Mol. Biol., Volume: 25, (1994), pp. 989-994

2. Hellens R.P., et al. "pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant

transformation", Plant Mol. Biol., Volume: 42, (2000), pp. 819-832 CrossRef

3. Odell J.T., et al. "Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S

promoter", Nature, Volume: 313, (1985), pp. 810-812

4. Gallie D.R., et al. "Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and

translation", Plant Cell, Volume: 1, (1989), pp. 301-311 CrossRef

5. Wesley S.V., et al. "Construct design for efficient, effective and high-throughput gene silencing in plants", Plant J.,

Volume: 27, (2001), pp. 581-590 CrossRef


30

Mutagenesi

Mutagenesi random

Error-prone PCR (EP-PCR) is the method of choice for introducing random mutations into a defined segment of DNA

that is too long to be chemically synthesized as a degenerate sequence. For shorter (100 bp) DNA segments, the most

powerful method is to chemically synthesize the sequence with a low degree of nucleotide degeneracy. This method,

however, is too expensive and time-consuming to be used for mutagenizing long stretches of DNA. Using EP-PCR, the

5‘ and 3‘ boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to

mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be

controlled as a function of the number of EP-PCR doublings performed.

The EP-PCR technique described here is based on the protocol of Cadwell and Joyce (1992). EP-PCR takes

advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the

addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.

After choosing a region of DNA to randomly mutagenize, one must decide on the desired level of mutagenesis that is

best suited to the project. If the mutation rate is too low, it may not be possible to find the potentially rare variants of

interest. If the mutation rate is too high, nearly all of the resulting library molecules will carry multiple mutations and

may therefore be inactive. The desired extent of mutation depends on the type of activity one is attempting to generate

and the number of library members that can be screened. For example, if one is attempting to generate an activity that

may require multiple mutations, such as a novel binding or catalytic activity, then it may be necessary to generate

molecules with several mutations per template. In this case it would also be important to have a screening or selection

protocol that would allow for the evaluation of a large number of variants. On the other hand, if one is looking for

mutations causing loss of protein function or decrease in thermal stability, for example, then 1 to 2 mutations per

template may be ideal. In this case it may be sufficient to screen a smaller number of variants.

The average number of mutations per template increases as a function of the number of doublings in the EP-

PCR reaction (Table 8.3.1). It should be noted that it is the number of doublings that is the determining factor, rather

than the number of EP-PCR cycles. Each cycle of EP-PCR generally increases the amount of DNA by a factor of 1.7 to

1.9 until the DNA concentration reaches a plateau and then stops increasing altogether. The point at which this plateau

occurs depends on the template and primer lengths and sequences, but is generally in the range of 5 to 50 ng/ul (1012-

1013 molecole). Table 8.3.1 Average Number of Mutations per DNA Template as a Function of Template Length and

Number of EP-PCR Doublings

EP-PCR doublings

Mutations per nucleotide

position

Template length

100 bp 200 bp 400 bp 800 bp 1600 bp

5 0.0033 0.33 0.66 1.3 2.6 5.3

10 0.0066 0.66 1.3 2.6 5.3 11

20 0.013 1.3 2.6 5.3 11 21

30 0.020 2.0 4.0 7.9 16 32

50 0.033 3.3 6.6 13 26 53

Prior to performing the actual EP-PCR reaction, it is important to run a pilot reaction to determine the amplification

efficiency under the EP-PCR reaction conditions. This should be evaluated for two reasons. First, if the amplification

per cycle is too low (1.7-fold increase in product DNA concentration per cycle), DNA fragments that contain one or

both of the primer-binding sites, but are shorter than the desired product, may have a strong selective advantage for

amplification. These shorter fragments are produced by mispriming during normal or error-prone PCR. After several

cycles, these shorter sequences may "take over" the EP-PCR reaction. This can be an especially severe problem when

many cycles (15) are to be performed. Second, the amplification per cycle must be known in order to calculate the

number of EP-PCR cycles necessary to achieve the desired number of doublings.

The amount of DNA amplification per EP-PCR cycle can be determined by diluting a known amount of the

unmutagenized PCR product, then amplifying it using the EP-PCR protocol and occasionally removing portions of the

reaction for quantitation on an ethidium bromide-stained agarose gel. The amplification per cycle should generally be

1.7. The yield per cycle can be optimized by altering the annealing temperature. Also, the extension time should last

for at least 3 min, to ensure complete extension. If this does not bring satisfactory results, longer or shorter primers may

have to be used. Primer lengths of 20 to 40 nucleotides usually produce acceptable results. Other parameters may also


31

be optimized to improve the amplification, (the Mg2+ concentration should not be reduced, however, as this may lead

to increased fidelity of DNA synthesis).

Table 8.3.1 outlines the average number of nucleotide substitutions per template as a function of the number of EP-PCR

doublings and the length of the template. Table 8.3.2 shows what fraction of the resulting products will be completely

free from mutation. Table 8.3.2 Fraction of Nonmutated DNA Templates as a Function of Template Length and

Number of EP-PCR Doublings

EP-PCR doublings

Mutations per nucleotide

position

Template length

100 bp 200 bp 400 bp 800 bp 1600 bp

5 0.0033 0.72 0.52 0.27 0.071 0.0050

10 0.0066 0.52 0.27 0.071 0.0050 2.5 x 10-5

20 0.013 0.26 0.070 0.0049 2.4 x 10-5 5.8 x 10-10

30 0.020 0.14 0.018 0.00033 1.1 x 10-7 1.3 x 10-14

50 0.033 0.035 0.0012 1.5 x 10-6 2.2 x 10-12 4.8 x 10-24

In most cases, the mutagenized DNA of interest will encode a protein. The fraction of mutated amino acids will be

higher than the fraction of mutated nucleotides by a factor of ~2.2. This is because a mutation in any of the three

positions of a codon may result in an amino acid substitution. If the initial template is a random open reading frame

(i.e., equal probability of each nucleotide at each position in each codon), mutation at the first position of a codon will

cause an amino acid change 96% of the time; mutation at the second and third positions will cause amino acid changes

100% and 23% of the time, respectively.

COMMENTARY

The simplest and most versatile method for introducing random point mutations into a DNA sequence is to take

advantage of the inherently low fidelity of Taq DNA polymerase. This enzyme can misincorporate with a frequency as

high as 0.02% per position. By performing exponential amplification through PCR, these errors will accumulate. Leung

et al. (1989) showed that the fidelity of copying by Taq polymerase can be further decreased by skewing the relative

dNTP concentrations, using a high Mg2+ concentration, and including Mn2+ in the reaction mixture. Cadwell and Joyce

(1992) used a similar strategy to obtain an error rate per nucleotide position of about 0.066% per PCR cycle. This

protocol, which forms the basis of that described here, has many useful qualities: (1) it is extremely simple in that it

does not require any unusual reagents such as nucleotide analogs; (2) the EP-PCR efficiency is comparable to that for

standard PCR conditions; (3) in many instances the desired level of mutagenesis can be obtained in ~1 hr with a single

PCR reaction; and (4) a wide spectrum of nucleotide substitutions is generated.

Critical Parameters

The EP-PCR protocol produces all types of substitution mutations, but the distribution is highly nonrandom. The resu

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