MBII - L7 - Gene Cloning 2 - Polymerase Chain Reaction

8/8/2019 MBII - L7 - Gene Cloning 2 - Polymerase Chain Reaction

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Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (PCR)

Lecture 7 [Page 1]

PCR allows us to make copies of DNA without cloning (time consuming process)

Enables production of many copies of a specified DNA sequence from a complex mixture, provided some

knowledge of the sequence is known

Method:

Exponential amplification occurs. After 30 cycles, template : new DNA will be 1 : 230(109 x)

Any biomaterial containing DNA can be used given appropriate preparation. Very little DNA is required.

Only a very small amount of the initial template remains, the majority of the DNA is newly synthesised.

Useful in forensics large amounts of DNA can be produced from a small sample

Advances in PCR:

Old Used E. coli DNA pol Klenow fragment. Was heat labile, so fresh enzyme had to be added after each

cycle. Time consuming and required lots of enzymes.


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Lecture 7 [Page 2]

New Taq polymerase used instead. A thermostable enzyme derived from Thermus aquaticus (a thermophilic

bacterium). This enzyme lasts for the whole reaction.

Old All reactions were carried out in 3 water baths, by hand. Time consuming

New Thermal cycler incorporated into a PCR machine

Conditions:


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Lecture 7 [Page 3]

Paternity testing Forensic medicine (who does this blood belong to) Is a patient infected with a certain pathogen

II) Amplification of genes prior to cloning

The DNA sequences for any genes are already in databases, so if you know what gene you want toclone then you can design a PCR primer for it.

The primers will amplify the DNA enough to allow you to clone the product The most common method for gene cloning provided the gene of study is known

1. Locate the cDNA sequence for the gene by looking in a database

2. Design primers to amplify this portion of DNA

3. Take a sample of mRNA for this gene

4. Carry out RT-PCR (Reverse Transcriptase PCR)* (B)

Primer 1 copies mRNA to cDNA Primer 1 + 2 amplify the cDNA

5. PCR products can now be cloned

*If using genomic DNA (A), no need for RT-PCR*

RT-PCR:


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Lecture 7 [Page 4]

1. cDNA step

Reverse transcriptase and dNTPs are added to purified mRNA Treat with alkali or RNase to destroy any RNA Use a polyT primer (will hybridise with the polyA tail of mRNA)

2. PCR step

Use random or specific primers (if sequence is known)The cDNA produced can then be cloned into vectors, used to measure mRNA content of a cell or tissue.

How is mRNA purified?

III) Colony screening of transformants

If you want to determine which colonies have a correct insert

1. Take DNA samples from several colonies

2. Carry out PCR using specific primers for insert of interest

3. Run products on a gel

4. Look for bands of a correct size

5. (always include a blank PCR experiment using water to ensure there is no DNA contamination)

IV) Adding and mutating DNA sequences by PCR

The primer sequence will always be incorporated into the final product, so if primers can be used that contain

a few additional / different bases.

The primer does not need to be 100% faithful, especially at the 5 end. The 3 end is the most important for

primer elongation.

This allows construction of altered DNA fragments.

We can add up to 20 bases at the 5 end

When introducing restriction enzyme sites, ensure 4 random bases are added each side of the re cognition

sequence. Some REs cannot function if there are not 4 bases surrounding their recognition sequence.

It is easy to change / add bases at the end of the target sequence.

Doing this in the middle is more difficult. 2 complimentary oligos are used, both cover the point of mutation.

The oligos contain the altered sequence.

A plasmid containing the gene is then denatured, and PCR is carried out. Pfu is used to ensure accuracy. A

full round of replication will result in an unchanged parental plasmid and a newly synthesised plasmid with

both strands containing the mutation. Dont religate yet!

The reaction product is then treated with Dpnl (a 4 base cutter). This will degrade methylated and hemi-

methylated DNA, so the parental DNA will be destroyed. The mutant DNA survives because Pfu does not

methylate DNA.


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Lecture 7 [Page 5]

The mutant DNA is then transformed into E. coli, which will naturally religate the nicks in the mutant DNA. The

fragments made by Dpnl will not be recovered.

Cells are then plated onto agar and selected to identify transformants

Colonies are picked.

This allows AAs to be introduced or changed modify proteins

We can then study the effect of the mutation, and see if protein function is changed (e.g. active sites)

V) Investigating gene expression

1. Isolate mRNAfrom normal and diseased tissue

2. Use RT-PCR to generate cDNA

3. Carry out PCR using primers for the gene that codes for the transcript

4. Run samples on an agarose gel

This will result in parallel bands (the PCR products)

Brighter / thicker bands indicate more mRNA > gene is being upregulated

This is semi-quantative

Quantative PCR (Real Time PCR):

Normal PCR does not allow exact quantification of template DNA

Monitoring the progress of the reaction allows measurement of the rate of reaction during the exponential

phase. This will be proportional to the amount of template.

The reaction is carried out as it normally would, but a third oligo primer is used that contains a fluorophore

and a quencher.

This oligo will bind to ssDNA within the region of amplification.

DNA synthesis will decouple the quencher, allowing fluorescence.


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Lecture 7 [Page 6]

This can be measured as an absorbance reading.

Intially, the fluorescence will be too low for accurate detection.

Absorbance must cross the threshold of detection in order to be accurate

Forensics:

Human DNA contains many repeats

One of these is known as microsatellite DNA (found at specific loci in the human genome)


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Lecture 7 [Page 7]

Capillary Electropheresis:

Agarose / polyacrylamide gels are difficult to automate and are unsuitable for high throughput analysis.

Instead, the fragments are run through a capillary, and the labelled products are detected by laser.

This technique has replaced gels for DNA sequence analysis

MBII - L7 - Gene Cloning 2 - Polymerase Chain Reaction

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