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

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:

Lecture 7 [Page 1]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (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.
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:

<100ng DNA (100 cells, 10-9g DNA)

dNTPs
Buffer
2 Primers (synthetic oligonucleotides)
 A pair of different primers are used, that will flank the sequence of interest
 17-30nt
 Avoid secondary structure
 No complimentarily between primers
 50% GC content is ideal. <<50% = difficult to anneal. >>50% = Anneals too strongly
MgCl2 – [Mg2+] is very important. Required for the action of DNA pol, but if there is too much it will
interfere with binding to dNTPs
Polymerase

1. Heat to 95C for 5 minutes (denatures strands)

2. 55C for 30 seconds (primers anneal)
3. 75C for 2 minutes (polymerase extends)
4. Repeat 1-3 0 times (30 cycles)
5. Run on agarose gel to check yield and size of amplified fragment

Taq polymerase is very efficient, but it lacks 3’5’ proofreading activity (its error rate is
1/103)
Pfu and Vent are thermostable polymerases with 3’5’ proofreading activity (more accurate)
 Synthesise fewer nts before reaction terminates and are expensive

As very small amounts of DNA will be amplified, contamination is a big risk.

False priming can occur where primers bind to different sites – Run on a gel, look for multiple
bands, if so then repeat. Smaller templates (10kb) have a reduced chance of false priming

Lecture 7 [Page 2]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (PCR)

Uses of PCR:
I) Identity – determine whether a particular fragment of DNA is present in a sample.
 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 to clone 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*

Lecture 7 [Page 3]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (PCR)

RT-PCR:

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 RE’s 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.

Lecture 7 [Page 4]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (PCR)

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. Don’t 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.
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 AA’s 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 mRNA from 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):

Lecture 7 [Page 5]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (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.
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)
 <6bp unit repeated 10-30 times
 Highly variable, due to mutation
 Each sequence contains a different number of repeats
 VNTRs – Variable Number of Tandem Repeats
 aka SSLP – Single Sequence Length Polymorphisms
 Can be used for genetic profiling

1/10,000,000,000 will have matching genetic patterns

Capillary

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

Lecture 7 [Page 6]
Molecular Biology II Gene Cloning 2: Polymerase Chain Reaction (PCR)

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

Lecture 7 [Page 7]