8. DNA AMPLIFICATION
Amplification means making multiple identical copies (replicates) of a DNA sequence. This can be carried out by various different methods, including cell cloning where host cells (manipulated using a vector to contain a DNA insert of interest) are allowed to divide and, as they do so, the insert is replicated also. However, one particular method of DNA amplification has proved very important in recombinant DNA technology and is used in a range of applications in medicine and forensic science. That method is PCR.
a) What is PCR?
PCR stands for the Polymerase Chain Reaction and was developed in 1987 by Kary Mullis and associates.
PCR is capable of producing enormous amplification (i.e. identical copies) of a short DNA sequence from a single molecule of starter DNA.
It is used to amplify a specific DNA (target) sequence lying between known positions (flanks) on a double-stranded (ds) DNA molecule.
The amplification process is mediated by oligonucleotide primers that, typically, are 20-30 nucleotides long. The primers are single-stranded (ss) DNA that have sequences complementary to the flanking regions of the target sequence. Primers anneal to the flanking regions by complementary-base pairing (G=C and A=T) using hydrogen bonding.
The amplified product is known as an amplicon.
Generally, PCR amplifies smallish DNA targets
100-1000 base pairs (bp) long.
(It is technically difficult to amplify targets >5000 bp long.)
PCR has many applications in research, medicine and forensic science.
b) How does it work?
- sample dsDNA with a target sequence
- thermostable DNA polymerase
- two oligonucleotide primers which are complementary to the sequence flanking the target sequence
- deoxynucleotide triphosphates (dNTPs)
- reaction buffer containing magnesium ions and other components
1. Heat denaturation
A DNA molecule carrying a target sequence is denatured by heat at 90-95oC. The two strands separate due to breakage of the hydrogen bonds holding them together.
2. Primer annealing
In the presence of an excess of dNTPs (the 'building blocks' of new DNA material), oligonucleotide primers are added. The primers are complementary to either end of the target sequence but lie on opposite strands. As the mixture cools at a lower temperature (50-65oC), each strand of DNA molecule becomes annealed with an oligonucleotide primer complementary to either end of the target sequence.
3. Primer extension
DNA polymerase is then added and complementary strands are synthesized at a temperature of 60-75oC. The polymerase causes synthesis of new material in the 5' to 3' direction away from each of the primers.
Following primer extension, the mixture is heated (again at 90-95oC) to denature the molecules and separate the strands and the cycle repeated.
Each new strand then acts as a template for the next cycle of synthesis. Thus amplification proceeds at an exponential (logarithmic) rate, i.e. amount of DNA produced doubles at each cycle.
30-35 cycles of amplification can yield around 1μg DNA of 2000bp length from 10-6μg original template DNA. This is a million-fold amplification!
(click for larger image)
Initially the 3 different stages at 3 different temperatures were carried out in separate water baths but nowadays a thermal cycler is used (a machine that automatically changes the temperature at the correct time for each of the stages and can be programmed to carry out a set number of cycles).
A typical thermal cycle might be as follows:
Heat denaturation at 94oC for 20 seconds
Primer annealing at 55oC for 20 seconds
Primer extension at 72oC for 30 seconds
Total time for one cycle = approx. 4 minutes (You can't simply add up the different times for the stages above because heating and cooling between each stage also have to be considered!!!)
Following PCR, the amplification product can be detected using gel electrophoresis where visualization of a band containing DNA fragments of a particular size can be indicate the presence of the target sequence in the original starter DNA sample. Similarly, absence of a band may indicate that the target sequence was not present in the original starter DNA sample. In this way, PCR can be used in combination with other techniques to not just simply amplify DNA (which, in essence, is all it does!) but also to detect specific target sequences.
PCR can be an extremely sensitive technique but is prone to contamination (unless scrupulous precautions are taken) leading to false positive results.
c) Fidelity and Taq DNA polymerase
Initially, DNA polymerase enzymes such as E.coli polymerase I (including Klenow fragment), and T4 polymerase were used for primer extension.
These DNA polymerases possess very high fidelity (accuracy of copying) due to proof-reading exonuclease activity (in 3'--->5' direction).
But two main drawbacks with these types of polymerase:
High temperature needed for DNA dissociation between cycles of amplification causes inactivation of the enzyme, so fresh enzyme needs to be added every cycle - very inconvenient and time consuming.
An important breakthrough was the use of thermostable DNA polymerases. These do not denature at the temperatures used to cause denaturation of the DNA and, therefore, fresh aliquots of enzyme do not have to be added after each cycle. This also meant that the entire process could more easily be automated.
The most well-known of these thermostable DNA
polymerases is Taq.
This enzyme has a molecular size of 94kD and an optimum reaction temperature of 75-80oC. But it is also stable at the higher temperature used for heat denaturation of the sample DNA (i.e. 90-95oC).
Taq polymerase comes from the bacterium Thermus aquaticus which lives in hot springs and would not survive in nature if it did not have special adaptations such as this thermostable DNA polymerase.
Taq allows oligonucleotide annealing and primer extension to occur at high temperatures without itself being denatured.
Advantages of Taq:
Disadvantage of Taq:
Other thermostable DNA polymerases:
61kD fragment of Taq polymerase but approximately two-times more thermostable and with optimal activity over a wider range of magnesium concentration.
Recombinant Taq polymerase
This has the advantage over 'natural' Taq enzyme of greater batch conformity and, hence, higher reproducibility.
2) Technical applications of PCR
a) Generation of probes
Cloned DNA can be amplified using primers complementary to known vector sequences flanking an insert
Also, uncloned genes can be amplified from 1st strand cDNA if portion of amino acid sequence at either end of protein product is already known.
b) Generation of cDNA libraries
Eukaryote mRNA has poly A tail at 3' terminus.
However, error rate of 0.25% means the accumulation of a significant number of errors.
Therefore, need to sequence several independently-isolated clones of a gene of interest to confirm correct sequence.
c) Production of DNA for sequencing
d) Analysis of mutations
Deletions and insertions in a gene can be detected by differences in size of amplified product.
Point mutations can be detected by using competitive nucleotide priming:
3) PCR in medicine and forensic science
a) Diagnosis of monogenic diseases (single gene disorders)
Since1987, PCR has had a major impact on pre-natal diagnosis of single gene disorders. PCR has also proved very important in carrier testing.
For pre-natal diagnosis, PCR is used to amplify DNA from foetal cells obtained from amniotic fluid.
Single base changes then detected by one or more of following:
Important to be certain of result so combination of two methods provides confirmation.
Other conditions which can be detected with the same approach include:
In DMD, affected gene is very large - 2Mb; codes for a cytoskeletal protein, dystrophin.
b) Detection of microorganisms
Generally, PCR and other nucleic acid-based methods such as probes are often faster, more specific and more sensitive than conventional methods. However, because nucleic acid-based methods are not available for all microorganisms, not appropriate for some, and too expensive for others, they tend to be used for:
Note that to use nucleic-acid-based methods to detect characterize and identify microorganisms, a DNA or RNA target sequence unique (or certainly very rare in other microorganisms) must be known in order to produce primers or probes complementary to it. Also, this sequence must be highly conserved, i.e. present in all/most strains and variants of the particular species. The 16S gene (which codes for one of the ribosomal sub-units) is often used as a target since the DNA sequence is generally unique to a particular species and it is highly conserved. Alternatively, the gene coding for an unusual phenotypic character (e.g. a biochemical reaction) can be targeted. For instance, the gene product (say an enzyme) could be amino-acid sequenced and then the DNA sequence coding for the product deduced by "Reverse Genetics" (i.e. working backwards from the protein product to the DNA sequence using our knowledge of the genetic code, codon usage, etc.). The process is called "Reverse Genetics" because it is the reverse of what happens in nature where the starting point is the DNA sequence and the product (after transcription and translation) is an amino acid sequence and, finally, a protein.
It should also be noted that most nucleic-acid-based methods cannot distinguish live (viable) cells from dead (non-viable) cells. This may not matter with some infectious diseases where the mere presence of the pathogen in a specimen indicates disease, e.g. syphilis (Treponema pallidum), but is not so useful with specimens where only the presence of viable (live) cells may be considered significant, e.g. Salmonella food poisoning species in food. However, in some cases live microbial cells can be detected by NA-based methods, e.g. by targeting microbial mRNA in a specimen. Since mRNA has a short half life compared to DNA, its presence in a specimen may indicate viable cells as its source.
Similarly, if NA-based methods such as PCR are used to detect a gene responsible for the pathogenicity of a microorganism (e.g. toxin production or antibiotic resistance) the mere presence of the gene is not necessarily indicative of the presence of the gene product since gene expression may not be occurring at the time. Again, though one could target the mRNA which is, at least, indicative of the initial stage of gene expression, i.e. transcription.
Rapid diagnosis may pre-date appearance of antibodies in blood of patient.
e.g. Mycobacterium tuberculosis
The bacterium that causes tuberculosis (TB) is conventionally identified by:
PCR can detect DNA target sequences diagnostic of Mycobacterium tuberculosis in a matter of hours. The saving in time over conventional methods means that patient treatment and tracing of contacts can begin much sooner.
c) PCR in forensic science
Crucial forensic evidence may often be present in very small quantities, e.g. one human hair, body fluid stain (blood, saliva, semen). Often there is too little material for direct DNA typing and other analyses. But PCR can generate sufficient DNA from a single cell!
PCR also possible on extensively degraded DNA. Examples include: DNA from single dried blood spot, saliva, semen, tissue from under fingernails, hair root, etc.
Other advantages of PCR in forensic science are:
Main legal problem with PCR is that identification is made from copied DNA rather than original material.
There are not only many variants of PCR, but also alternative amplification techniques in use. For instance, amplification can be carried out by the use of a host-vector system and cell cloning. Before the development of PCR, this was the main method used but it is not as powerful nor as convenient for most purposes as PCR.
Another example of an alternative amplification technique is the Ligase Chain Reaction (LCR).
This is a DNA amplification technique which can be used to detect trace levels of known nucleic acid sequences. LCR involves a cyclic two-step reaction:
The products of the ligation from one cycle serve as templates for the next cycle’s ligation reaction. LCR results in the exponential amplification of the ligation products in a manner analogous to the exponential amplification of template in the PCR reaction. An example of an application of LCR is the detection of DNA sequences specific to particular microorganisms (e.g. Chlamydia trachomatis) to aid identification and diagnosis of the disease caused.
An example of a variant of PCR used to detect mutations is the Amplification Refractory Mutation System (ARMS).
Also known as: Allele Specific PCR (ASPCR); PCR Amplification of Specific Alleles (PASA).
This is an amplification technique used for the detection of known single-base substitutions or microdeletions/insertions. Two complementary reactions are used. One contains a primer specific for the normal allele and the other reaction contains a primer for the mutant allele (both have a common 2nd primer). One PCR primer perfectly matches one allelic variant of the target but is mismatched to the other. The mismatch is located at/near the 3' end of the primer leading to preferential amplification of the perfectly matched allele. Genotyping is based on whether there is amplification in one or in both reactions. A band in the normal reaction only indicates a normal allele. A band in the mutant reaction only indicates a mutant allele. Bands in both reactions indicate a heterozygote. ARMS can detect a mutant allele in the presence of 40 copies of the normal allele. ARMS is claimed to be: rapid (1 working day), reproducible, inexpensive, automatable.
ARMS can be used to screen for homozygous and
heterozygous (carrier) states for: cystic fibrosis, alpha-1-antitrypsin
deficiency, sickle-cell anaemia, phenylketonuria, apolipoprotein E, B-thalassaemia,
Suggested further reading:
Brown, T.A. (2001).
Gene Cloning & DNA Analysis. (4th edition).
Chapter 9 is devoted to PCR.
Primrose, S.B. et al. (2001).
Principles of Gene Manipulation.
for you to think about
(after you have read this section and consulted a textbook)
1) Describe in your own words what PCR is and how it works.
What do you understand by the terms: (a) fidelity (b) proofreading?
2a) List the principle advantage and disadvantage of using E. coli polymerase I for PCR.
b) Why is Taq DNA polymerase better?
c) What is the main drawback with this enzyme?
3a) What is the main advantage in using PCR for gene sequencing?
b) Are there any problems with this approach? If so, what, and how would you overcome them?
c) Describe briefly how you would go about generating a series of deletion mutations in a cloned gene. Assume you have the sequence and access to an oligonucleotide synthesizer ("Gene Machine").
4a) List some of the inherited diseases which can be detected by PCR.
b) Why is it so important to get results as quickly as possible when undertaking pre-natal diagnosis?
c) What are retro-viruses? Why is it impossible to detect them by direct hybridisation?
5a) Describe some of the applications of PCR in forensic science.
b) What safeguards are necessary in obtaining evidence this way?
6) Describe what materials you would need to create Jurassic Park. How would you go about doing it?
Geoff Stubbs's notes on PCR and the practical protocol can be accessed by clicking here.
END OF SECTION
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