GENE CLONING TOOLS

Gene Cloning allows the separation and identification of a specific section of genetic material (DNA or RNA) from other sequences. It then allows the isolation of large numbers of copies of this sequence for molecular characterisation

Genetic engineering

Other terms that you will see that mean the same thing include:

DNA cloningmolecular cloning

recombinant DNA technology

What is a gene and what is a coding region?

A gene is a nucleic acid sequence that code for a polypeptide or chain that has a

function in an organism

A gene sequence includes regulatory regions that are responsible for controlling

the spatial and temporal expression of the gene product (a protein or RNA)

A protein is encoded by a coding region which is the part of the gene between the

translation initiation codon (normally ATG)and the translation termination codon

(TAA, TGA or TAG)

It is important that you appreciate the difference between a gene and a coding

region.

In many genetic engineering experiments we will wish to express a protein and so

will only be interested in the coding region, not in the remainder of the gene from

which it is derived.

Some uses of genetic engineering

Cloning allows full characterisation of a gene including identification and analysis of regulatory sequences and mechanisms controlling spatial and temporal gene expression (i.e. when and where the gene is expressed) by;

DNA sequence analysis

Determination of 5' and 3' ends of the mRNA transcript

Location of introns/exons,

Analysis of mutated forms of DNA

Transcription control elements

Trans-factors

Sequences that control transcript stability

Localisation of expressed protein

Reverse genetics

2. Genome mapping and evolutionary studies

3. Expression of recombinant proteins, from the coding region, for structural and functional studies or large scale production of industrial or medical proteins

3. Protein engineering and directed evolution to generate new functional proteins

4. Diagnosis of human genetic diseases/ Forensic analysis

5. Gene therapy

6. Transgenic plants and animals

Some uses of genetic engineering

We use a range of enzymes as basic tools to manipulate DNA and RNA during gene cloning and analysis processes

Restriction enzymes (site-specific cutting)

Phosphatases (removing 5’ phosphates)

Kinases (adding 5’ phosphates)

Ligases (joining fragments)

Nucleases (removing DNA)

Oligonucleotides (synthetic DNA eg primers and probes)

DNA polymerases (replicating, amplifying) eg. DNA sequencing, PCR, mutagenesis

Tools and techniques

We use various approaches to investigate genes, gene expression and to characterise where and when a gene is expressed, and

where and when its product is localised and active.

Gene Cloning: Vectors, enzymes, PCR, agarose gelsGenes and polymorphisms: Southern blot, DNA

sequencing, Next generation sequencingTranscript analysis: Northern blot, intron/exon, start site

mapping, in situ hybridisationGlobal analysis: microarrays, proteomics, transgenic

knockout/in, Next generation sequencingProtein expression profiles: western blot,

immunocytochemistry, GFP, fusion proteinsProtein expression studies: over-expression, functional analysis

in cellsMolecular interactions: immunoprecipitation, phage display,

yeast hybrid systems, FRET, SPR,

Tools and techniques

Catalase coding region

?

Experimental design sub-cloning

How can I sub-clone this catalase coding region into an

protein expression vector so that I can express and purify the

catalase?

What steps would I need to follow and what tools/techniques

would I need to use?

Let’s think about what tools are available.

Non-coding regions

1. Vectors

2. Agarose gels

3. Restriction enzymes: cut DNA

4. Modifying enzymes: remove or add chemical group (eg

phosphate or nucleotide)

5. Ligases: join DNA

6. Polymerases: synthesise DNA (& RNA) and/or remove

nucleotides

7. Synthetic DNA – oligonucleotides, synthetic genes

8. Polymerase chain reaction PCR

Key molecular biology tools

Now let’s consider a basic gene cloning flow diagram

Basic Steps in CloningPurify vector DNA (e.g.

plasmid or phage)

Alkaline lysis

Purify target DNA to

be cloned eg

genomic, cDNA, or in

silico sourced clone

Transform ligation mix

into competent E. coli.

One cell takes up one

DNA molecule

Plate onto agar with

antibiotic

Only plasmid

containing cells grow

Colonies form on

plates by cell growth &

plasmid replication to

give a clone

Screen colonies to identify

those with recombinant

plasmid

Colony PCR or plasmid

isolation & restriction digest

Digest the circular

plasmid DNA with

Restriction enzyme(s)

Digest the target DNA

to be cloned with

Restriction enzyme(s)PCR amplify a DNA

fragment with

carefully designed

primers & digestLigate (join) digested

vector and target DNA

Mixture of vector &

recombinants

Alakaline phosphatase

treat the plasmid DNA

to remove 5’ P’s

First I need to prepare DNA for cloning

http://www.acgtinc.com/

http://www.pharmatech.co.kr/

http://www.acgtinc.com/

PLASMID

DNAcDNA

GENOMIC

DNA

/oligo dT

purification of

mRNA

First you need to prepare DNA for cloning

http://www.acgtinc.com/

cDNA

/oligo dT purification

of mRNA

We are going to recover the catalase coding region from cDNA that is synthesised

from mRNA.

The mRNA must be isolated from the correct cells and purified (ca. 3-5%) from other

RNAs

This is done by using an oligo dT column or oligo dT magnetic beads to isolated

mRNA which is polyadenylated.

cDNA synthesis then relies upon the enzyme Reverse transcriptase and a primer,

usually an oligo dT primer for first strand synthesis and then a self-priming or

specific primer plus a DNA polymerase for second strand synthesis.

If we know the gene sequences we can actually design two primers that are specific

for the coding region for use in first strand cDNA synthesis followed by PCR

Let’s assume that we are starting with a collection of oligodT-

primed cDNA molecules; some of these will be ones that

contain our catalase sequence

Catalase coding region

We know the sequence of the gene from genome sequencing projects and can

access this information from databases such as Genbank

So we can design primers that can be used for PCR amplification of only the

coding region of the cDNA

Polymerase Chain Reaction

http://www.youtube.com/watch?v=eEcy9k_KsDI

Thermostable DNA polymerases:

DNA synthesis at high temperatures in PCR and other reactions

Taq• 5’ to 3’ exonuclease

and 5’ to 3’ DNA synthesis

Kod, Pfu• 5’ to 3’ DNA synthesis

and 3’ to 5’ exonuclease (proof-reading)

72 Co

94 Co

55 Co

etc

Initial

Denaturation Cycle 1 Cycle 2

Denaturation

Time

Te

mp

era

tu

re

Annealing Extension

PCR involves thermal cycling – 25-40 cycles

5'

3'

template

primer

Eg. Restriction enzyme site

Promoter sequence eg T7 DNA pol

can be added to the 5’ end

• About 20 nt long primers

• ~50 % GC if possible and with similar TM >55 oC

• Avoid complementary primer sequences

• Avoid polypyrimidine (T, C) or polypurine (A, G) stretches

• Can add sequences to 5’-end

Things to consider in PCR primer design

How do we design primers for PCR?

We know the sequence of the gene from genome sequencing projects and can

access this information from databases such as Genbank

So we can design primers that can be used for PCR amplification of only the

coding region of the cDNA

But….how will we be able to clone this PCR amplified coding sequence into a

cloning vector?

First we need to decide what cloning vector we will use

5’ 3’

5’3’

ATG

TAA

TGA

TAG

Plasmids

Viruses/Bacteriophage

Cosmids• combination of plasmid and bacteriophage l

Phagemids– combination of plasmid and bacteriophage M13

Common cloning vectors

pET28 TEV

5368 bp

expression region

lacI

Kanr

His6 tag

TEV cleavage site

His6 tag

T7 P

f1 ori

pBR322 ori

T7 TERM

BamHI (5167)

BglII (4965)

ClaI (1251)

EcoRI (5173)

EcoRV (3797)

HindIII (5192)

KpnI (5112)

NcoI (5070)

NheI (5135)

NotI (5199)

SacI (5183)

SalI (5186)

SmaI (1070)

SphI (4776)

XbaI (5031)

XhoI (5207)

XmaI (1068)

An example of a

cloning vector used

routinely in my lab

Antibiotic resistance

kanamycin

resistance

Origin of replication

Expressed gene regulation

Promoter

Multiple cloning site

Purification of

expressed protein

pET28 plasmid

vector

Designing the primers for PCR?

If we are going to clone into pET28 we will need to add restriction sites at the

ends of the coding region

We do this by adding the restriction enzyme cleavage sequences to the 5’ end

of the primers.

If we add different sites to the 5’ and 3’ end of the coding region then we can do

directional cloning so that we know the sequence is inserted into the vector in

the correct orientation.

5’ 3’

5’3’

We will add an NcoI site at the 5’ end of coding region and EcoRI site at the 3’ end

EcoRI

When we PCR amplify the coding region using these primers we will

generate this sequence

We need to check whether we have the correct PCR product and digested vector

CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC

GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG

5’ 3’

5’3’

Always label 5’ and 3’ ENDS when writing

CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGACACACCTGCTAACACTC

GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTGTGTGGACGATTGTGAG

5’ 3’

5’3’

Select primer sites (ca. 20 nt)

A more detailed look at how to design the primers for

PCR of the coding region

CATCTGCTAGTCCAACCTACATCATGTCGTCAAGTCAT-1kb-ATTATTATCTCTCTGGATGTCAACATGAAACACCTGCTAACACTC

GTAGACGATCAGGTTGGATGTAGTACAGCAGTTCAGTA-1kb-TAATAATAGAGAGACCTACAGTTGTACTTTGTGGACGATTGTGAG

5’ 3’

5’3’

Add sequences onto primer with a few extra 5’ nucleotides to ensure efficient

restriction enzyme cleavage.

NcoI = CCATGG; EcoRI = GAATTC

NcoI EcoRI

PCR

3’GTCCAACCTACATCATGTCG 3’ GACCTACAGTTGTACTTTGT

Always write primers as 5’ to 3’

sequences so the reverse strand needs

rewritten TCGTCTTAAGTGTTTCATGTTGACATCCAG5’ 3’

NcoI

CCATGG5’

4 nts

ATCC

EcoRI

GAATTC 5’

4 nts

TGCT

• Electrophoresis through an agarose gel matrix

• At neutral pH DNA and RNA have a net NEGATIVEcharge due to phosphate groups and so move towards the ANODE (+ve electrode)

• Small molecules move through faster than longer/larger molecules so separation is on the basis of size

– For linear fragments rate of migration proportional to log10 molecular size

Can also separate on basis of conformationPlasmid DNA: Supercoiled, open circular

and linear are all the same molecular size but migrate differently

Analysing DNA fragments by agarose gel electrophoresis

pET28 TEV

5368 bp

expression region

lacI

Kanr

His6 tag

TEV cleavage site

His6 tag

T7 P

f1 ori

pBR322 ori

T7 TERM

BamHI (5167)

BglII (4965)

ClaI (1251)

EcoRI (5173)

EcoRV (3797)

HindIII (5192)

KpnI (5112)

NcoI (5070)

NheI (5135)

NotI (5199)

SacI (5183)

SalI (5186)

SmaI (1070)

SphI (4776)

XbaI (5031)

XhoI (5207)

XmaI (1068)

Next we need to restriction

digest the vector and PCR

product with NcoI and EcoRI

Endonucleases: Digest DNA at internal (often palindromic)

sites in DNA

– Restriction enzymes cleave DNA only at specific

recognition sites

– generating fragments for cloning

– map genes and polymorphisms (SNP’s)

5’3’

3’5’GAATTC

CTTAAG

5’3’

3’5’GAATTC

CTTAAG

5’3’

3’5’G3’

CTTAA5’

5’AATTC

3’G

Restriction enzymes

Animation: Restriction enzymes

• http://highered.mcgraw-

hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites

/dl/free/0072437316/120078/bio37.swf::Restriction

Endonucleases

Restriction enzyme sites

• Restriction enzymes can leave three different types of ends

– 5’ overhang (sticky end)

– 3’ overhang (sticky end)

– blunt

GAATTCCTTAAG

CAGCTGGTCGAC

GGTACCCCATGG

Eco RI Pvu II Kpn I

G3’ 5’AATTC

CTTAA5’ 3’G

CAG3’ 5’CTG

GTC5’ 3’GACGGTAC3’ 5’C

C5’ 3’CATGG

5’ OVERHANG BLUNT END 3’ OVERHANG

The ends generated allow different DNA fragments to be joined

Restriction enzyme sites

• Some enzymes recognise different sites but generate the

SAME sticky ends

GGATCCCCTAGG

Bam HI Bgl II Sau 3A

AGATCTTCTAGA

NGATCNNCTAGN

G3’

CCTAG5’5’GATCT

3’A+ GGATCT

CCTAGA

Will not cut with Bam HI

or Bgl II, but will still cut

with Sau 3A

Bam HI end Bgl II end Product

• Alkaline phosphatase:

– removes the 5’ phosphate groups from DNA, normally

the vector DNA

– needs inactivated usually by heat before the ligation

step (otherwise it can dephosphorylate the insert as

well!!)

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.

Hydrolysis of phosphate ester

+ 2 PO4

-3

Often the restriction digested vector DNA is also treated with the

enzyme

P

Why do we use alkaline phosphatase?

3’OH 5’PGATC

CATG5’ 3’OH

GATC

CATG

Vector Insert

Ligation

Vector plus insert

GATC

CATG

No ligation

3’OH 5’OH GATC

CATG5’OH 3’OH

Vector Vector

Vector with NO insert

• Generates phosphodiester bonds between 3’OH and 5’ P

• Must have a 5’Phosphate (5’P) for ligase to function

The vector and insert DNA are then mixed in a ligase buffer

containing ATP and DNA ligase

3’OH 5’PGATC

CATGP5’ 3’OH

GATC

CATG

Vector Insert

Join two double strand

molecules together if they

have suitable ends

3’OH 5’P

O-P-O

Repair single-strand breaks

in the phosphodiester

backbone useful in some site-directed

mutagenesis applications

The E. coli cells are treated with CaCl2 or

RbCl2 to disrupt their cell walls and can

be stored frozen at -80oC.

For a transformation reaction aliquots of

cells are thawed on ice and DNA is

added, typically around 40 -5 0 ng.

After incubating on ice the cells are heat

shocked for around 1-2 min at 42oC so

that cells take up the DNA

Very few of the cells will actually become

transformed and so we need to be able to

identify those cells that have been

transformed and we do this by antibiotic

selection

Following a ligation reaction an aliquot is

transformed into competent E. coli cells.

All the transformed

colonies will contain a

vactor, but NOT all will

contain recombinant

plasmids

(1) Clones containing vector molecules can grow –

they are antibiotic resistant!

• Reduce vector only (eg alkaline

phosphatase)

(2) How do you identify recombinants?

Blue white selection (based on lacZ activity)

Colony PCR

Purify plasmid & restriction digest

Hybridization screening

Most common

then

DNA sequence

Selection and screening

E. coli DNA polymerase I: synthesises DNA using a template and primer

– Three activities: • 5’-3’ exonuclease (repair)

• 5’-3’ DNA synthesis

• 3’-5’ exonuclease (proof-reading)

DNA PolymerasesUses: DNA synthesis (and sometimes as an

exonuclease) DNA sequencing DNA mutagenesis

DNA labelling

These are useful for some DNA manipulation including• Filling in sticky ends to make them blunt ends • Radioactive labelling ends• DNA synthesis reactions that use a primer

Klenow fragment

T4 DNA pol

T7 DNA pol

DNA Polymerases that are now more

commonly used than Pol I

• T7 and T4 phage DNA polymerases: – Klenow activities, but more efficient

• Thermostable DNA polymerases: – DNA synthesis at high temperatures in PCR and

other reactions

– Taq• 5’ to 3’ flap exonuclease and 5’ to 3’ DNA synthesis

– Kod, Pfu• 5’ to 3’ DNA synthesis and 3’ to 5’ exonuclease (proof-

reading)

• Reverse transcriptase: – synthesises cDNA using RNA as a template and a

DNA primer

1. Gene cloning essentials 3

1.1 Introduction 4

1.2 Gene cloning applications 4

1.3 Gene cloning in the laboratory 5

1.4 Gene cloning processes 14

1.5 Further types of gene cloning 18

1.6 Chapter summary 21

2. Polymerase chain reaction 23

2.1 Introduction 23

2.2 How PCR works 24

2.3 The PCR protocol 26

2.4 PCR techniques and applications 31

2.5 Forensic DNA analysis 40

2.6 Future prospects 41

2.7 Chapter summary 41

Reading associated with this lecture