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Objectives

• describe the steps in gene cloning by using plasmid as the vector

The steps for cloning:

1) Isolation

2) Splicing

3) Insertion

4) Transformation

5) Screening

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1. Isolation of DNA (gene)

Steps in Gene Cloning

2. Slicing with restriction enzymes

3. Insertion

4. Transformation & amplification (multiplication @ cloning)

5. Screening ; to identify bacterial cell containing recombinant plasmid Probing ; to identify bacterial cell containing recombinant plasmid with target gene

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isolation of plasmid DNA (from E. coli) and DNA containing gene of interest

- plasmid DNA as cloning vector

Isolation

♫ plasmid DNA carries ampR gene and lacZ gene

- ampR gene: antibiotic resistance gene - lacZ gene : encode for β-galactosidase

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Isolation

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cut open the plasmid DNA at restriction site which lies within lacZ gene

cut out the DNA into many DNA fragments; some of these fragments carry the gene of interest

- by using same restriction enzyme which cut at restriction sites containing same palindromic sequence to produce sticky ends

Slicing (cutting / cleave)

Insertion

after mixing, the DNA fragments and the cut plasmids form the complementary

pairs

they are then joined by DNA ligase

creating a mixture of rDNA molecules

♦ note that the lacZ has becomenonfunctional

♦ cannot codefor β-galactosidase

Transformation

bacteria containing recombinant plasmid CAN’T produce β-galactosidase

the rDNA are reintroduced into the bacteria

bacterial cells are mixed with rDNAin the presence of cold calcium chloride

followed by heating; making the bacterial cell wall permeable to plasmids

Transformation

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Transformation

produce diverse of bacteria :

• bacteria with recombinant plasmid (containing gene of interest) e.g. gene encode for insulin

• bacteria with recombinant plasmid (containing gene which encode for other protein) e.g. gene encode for human growth hormone

• bacteria with NON-recombinant plasmid

Screening

i.e. detecting the bacteria containing rDNA

Procedure 1 Blue-white screening

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- to identify bacteria containing recombinant plasmid

Blue-white screening

bacteria is cultured on medium containing antibiotic (ampicillin) and sugar (X-gal)

Observation

ONLY bacteria with plasmid grow ; has ampicillin resistance (ampR) gene

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X-gal is used to identify colonies bacteria with recombinant plasmids

bacteria colonies WITHOUT recombinant plasmid will stain blue ; β-galactosidase is produced by functional lacZ gene (hydrolyze X-gal to yield blue product)

bacteria colonies WITH recombinant plasmid will stain white ; β-galactosidase is NOT produced because lacZ gene is NON-functional; X-gal is NOT hydrolyzed

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Probing (Nucleic acid hybridization)

- to identify bacteria with recombinant plasmid containing gene of interest (target gene)

Probing (Nucleic acid hybridization)

based on base pairing between gene of interest (e.g. gene encode for insulin) and other DNA molecule known as DNA probe (short & single-stranded; labeled with radioactive isotope or fluorescent tag)

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Probing (Nucleic acid hybridization)

a master plate is prepared

STEP 1

- contain colonies of bacteria with recombinant plasmid in the mean time, DNA probe is prepared

STEP 2

nitrocellulose filter paper is placed onto the master plate the filter paper is pressed against the bacterial colonies on the master plate ; bacterial colonies is transferred onto the filter paper

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the filter paper is treated with NaOH or heat - to denature (separate) the DNA; double helix DNA single stranded DNA

STEP 3

Probing (Nucleic acid hybridization)

STEP 4

DNA probe solution is added to the filter paper

- DNA probe will hybridize (base-pair with any complementary bases of single stranded DNA)

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the filter paper is washed to remove the excess, unbound DNA probe

STEP 5

then, the filter paper is laid on X-ray film – autoradiography technique

the film (autoradiograph) is developed

Probing (Nucleic acid hybridization)

STEP 6

the autoradiograph is compared with the master plate the colonies which contain gene of interest is identified

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REMIND AGAIN

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Isolation

Slicing

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Insertion

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Transformation Screening

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Master plate

Probing

STEP 1

DNA probe

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Probing STEP 2

Contain NAOH or Heat

To denature DNA ,double to single

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Probing STEP 4

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Probing

STEP 5 STEP 6

The steps for cloning:

1) Isolation

2) Splicing

3) Insertion

4) Transformation

5) Screening

Screening

i.e. detecting the bacteria containing rDNA

Blue-white screening

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Probing (Nucleic acid hybridization)

- to identify bacteria with recombinant plasmid containing gene of interest (target gene)