MIC 210

BASIC MOLECULAR BIOLOGY

LECTURE 4

DNA CLONING

BY

SITI NORAZURA JAMAL (MISS AZURA)

03 006/ 06 483 2132

Outline

1. Source of DNA

2. Vector

3. Restriction enzyme

4. Ligation

5. Bacteria host

6. Transformation

7. Selection of recombinants

INTRODUCTION TO DNA

CLONING

What Does It Mean: “To Clone”?

Clone: a collection of molecules or cells, all identical to an

original molecule or cell

• To "clone a gene" is to make many copies of it - for

example, by replicating it in a culture of bacteria.

• Cloned gene can be a normal copy of a gene (= “wild

type”).

• Cloned gene can be an altered version of a gene (=

“mutant”).

• Recombinant DNA technology makes manipulating

genes possible.

• To work directly with specific genes, scientists prepare

gene-sized pieces of DNA in identical copies, a process

called DNA cloning

Fig. 20-2

DNA of chromosome

Cell containing gene of interest

Gene inserted into plasmid

Plasmid put into bacterial cell

Recombinant DNA (plasmid)

Recombinant bacterium

Bacterial chromosome

Bacterium

Gene of interest

Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest

Plasmid

Gene of Interest

Protein expressed by gene of interest

Basic research and various applications

Copies of gene Protein harvested

Basic

research on gene

Basic research on protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolves blood clots in heart attack therapy

Human growth hor- mone treats stunted growth

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• A preview of gene cloning and some uses of cloned genes

• Most methods for cloning pieces of DNA in the laboratory share

general features, such as the use of bacteria and their plasmids

• Plasmids

are small circular DNA molecules that replicate separately from the

bacterial chromosome

• Cloned genes are useful for making copies of a particular gene

and producing a protein product

• Gene cloning involves using bacteria to make multiple copies of a gene

• Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell

• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA

• This results in the production of multiple copies of a single gene

Gene cloning, genetic engineering,

recombinant DNA technology

They‟re more or less the same

It basically means :

joining together DNA from

different sources/organisms,

forming a recombinant DNA

molecule

Then put this recombinant DNA

into a host cell, usually bacteria

The host cell will then replicate

many copies of this recombinant

DNA molecule

Sometimes, we might want to

ask the host cell to use the

genetic information in the

recombinant DNA to make

proteins

Why genetic engineering ?

Medical & health

applications

Production of novel and

important proteins

Insulin.. See chapter 1

Agricultural applications

e.g. GM crops

„golden rice‟ - Inserting the gene

for synthesis of carotene

(Vitamin A) into rice

Cloning genes for scientific studies

Basic of DNA Cloning

The basics of cloning

You need :

1) Source of DNA - to be cloned

2) Choice of vectors – to carry,

maintain and replicate cloned gene in host

cell

3) Restriction enzymes - to cut DNA

4) DNA ligase - to join foreign and

vector DNA recombinant DNA

5) Host cell – in which the recombinant

DNA can replicate

1) Source of DNA

• Genomic DNA

– DNA extracted from cells and purified

• cDNA

– by reverse transcription of mRNA

• Amplified DNA

– using Polymerase Chain Reaction

• Synthetic DNA

– DNA made artificially using a machine

2) Vector

• to carry the ligated foreign gene into the host cell

• maintain the foreign gene in the host cell

• Replicate

• pass into new cells during cell division

• Expressed the cloned foreign gene to make a

protein

Different types of cloning vectors

•plasmids

•bacteriophage l, M13

•Cosmids, phagemids

•Artificial chromosomes

BAC, YAC, MAC etc.

Plasmid

• Extrachromosomal DNA found in

bacteria & fungi

• Close circular DNA molecules,

supercoiled

• Can replicate autonomously,

independent of chromosome

• Can be transfer to other cells by

conjugation

• Can be integrated into the

chromosome

• In nature, plasmids carry genes that are not essential under normal conditions

• But confers a survival advantage under extreme conditions eg. resistance to

antibiotics, metabolism of unusual substrates

• Number of plasmid per cell - controlled by plasmid itself

High copy number > 100 /cell; low copy number < 20 /cell

• Plasmid incompatibility – the presence of one plasmid in a cell excludes other

plasmids

pBR322 – a high copy number plasmid

Important DNA elements :

1. The rop (or sometimes ori)

origin of replication, so that the

plasmid can be maintained &

replicated in the host cell

2. Antibiotic resistance marker

genes (ApR for ampicillin

resistance and TcR for

tetracycline) so that we can

select

3. Unique restrcition sites (EcoRI,

PvuI etc) so that we can cut the

plasmid in one place only.

and insert the foreign gene we

want to clone

3) Restriction enzyme

> Type II Restriction endonuclease

• Enzymes found in some microorganisms

• Natural role to destroy invading foreign DNA – eg. bacteriophage DNA

• Recognizes very specific short sequences of DNA – Each enzyme has its own recognition sequence/ site – Sometimes two different enzymes have the same recognition

sites, in which case they are known as isoschizomers

• Cuts DNA in very specific manner

• Technically – one Unit of RE will completely digest 1 ug of substrate DNA in a 50 ul reaction volume in 60 minutes

Restriction enzymes cut DNA at very specific sequences

• HindIII PstI

• EcoRI FatI

• SexAI SspI

Recognition sites – always palindromic

-Formation of hairpin loops

How REs cut DNA

Sticky ends can re-anneal by base-pairing

Sticky ends has complementary overhangs

- allows for proper reannealing and joining of DNA molecules

Bacterial transformation

Inserting the recombinant DNA molecule into a Competent E.coli cell

The cells must be made competent be treating with CaCl2 or very little

DNA will be taken up.

Selecting for transformants carrying recombinant DNA

No vector or recombinant DNA

– will not grow on media + ampicillin

Vector only

– will grow on media + ampicillin

Recombinant DNA (vector + insert) –

will grow on ampicillin

This is the one we want !

The goal of any cloning experiment is to obtain transformants carrying

cloned insert DNA. There are several strategies to maximise these

Use two different restriction enzymes to cut each end of the vector

(and also the foreign DNA you want to clone)

- Generate different sticky ends – cannot self ligate

EcoRI BamHI

EcoRI BamHI

The goal of any cloning experiment is to obtain transformants carrying

cloned insert DNA.

There are several strategies to maximise these

1. Directional cloning

3. Dephosphorylation of

vector

-both the 3‟OH group and

5‟PO4 group are required for

ligation

-if the 5‟PO4 groups on the

vector ends are removed –

cannot self-ligate

-Using a phosphatase

enzyme

-e.g calf intestinal

phosphatase etc.

P

P

Blue white selection – lacZ complementation

The vector contains a portion of the E.coli LacZ gene.

A multiple cloning site (MCS) sequence is inserted into the LacZ‟ fragment

The LacZ gene codes for the b-galactosidase enzyme

The b-gal enzyme

hydrolyses lactose into

glucose and galactose

The LacZ gene can be broken into two parts, a and b

- each part encoding a fragment of the b-galactosidase enzyme

Inserted into

plasmid vector LacZa

LacZb’

b- fragment

A fully active enzyme can be reconstituted from both fragments

Inserted into

plasmid vector LacZa

LacZb’

b- fragment

The b-gal enzyme can

also hydrolyse a colorless

substance called X-Gal

into glucose and a blue

color pigment

To do blue white selection, the gene of interest is cloned into the MCS

Gene you

want to clone

Transformants are plated onto a medium containing :

o Antibiotic for selection

o IPTG to induce expression of the LacZ’

o X-Gal to detect the presence of b-galactosidase

Transformants with vector only :

o LacZ is expressed a fragment is produced

o Complements b-fragment to form fully active enzyme

o Hydrolyses X-Gal Blue color colonies

Transformants with recombinant DNA:

o LacZ is destroyed by insertion of foreign gene no a fragment

o Cannot form fully active enzyme

o No hydrolysis of X-Gal White color colonies

Just to remind you the basic steps….

Sometimes, a simple cloning vector is not good enough

We might want to ask the bacteria cell to make proteins using

information on the cloned gene

We need to use an expression vector

Expression vector

- clone foreign gene AND make foreign

protein

- requires extra DNA elements

Promoter – to initiate transcription –

synthesis of mRNA

Terminator – to stop transcription

Fusion tags – for making fusion proteins

e.g. Histidinex6, c-myc, HA, GFP

In frame MCS

Other things – e.g. Poly-A sites

Recombinant Insulin – not as easy as it looks

The insulin molecule as coded by DNA

Active insulin molecule

C-peptide is removed

Disulfide bonds formed between Peptide A & B

Not done by bacterial cell !

Production of recombinant insulin – „Humulin‟ in E.coli

DNA for peptide A and Peptide B – synthesized chemically

Peptide A – 21 amino acids – 63 nucleotides + ATG + stop codon

Peptide B – 30 amino acids – 90nucleotides +ATG +stop codon

Clone into a different plasmid vector s– into the gene for B-galactosidase

Both DNA‟s were cloned in frame with the b-gal gene

Expressed as fusion proteins – Peptide (A or B) + part of b-gal

This is necessary – otherwise the small peptides will be quickly degraded

Fusion with b-gal stabilises the peptides

Expression driven by the LacZ promoter

Fusion proteins are purified from the cells

The B-gal part is then cleaved off by reacting with cyanogen bromide

which cleaves methionine

The peptide and then purified and chemically reacted to form disulfide bonds

What is the problem of this approach ?