1 Objectives • describe the steps in gene cloning by u plasmid as the vector
Jan 21, 2016
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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)