Download DNA amplification 2

8. DNA AMPLIFICATION
1) Introduction
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 complementarybase 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?
Requirements:
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thermal cycler
PCR amplification mix typically containing:
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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
3 stages:
1.
Heat denaturation
A DNA molecule carrying a target sequence is denatured by heat at 9095oC. 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.
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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!
PCR DIAGRAMS
(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:
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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:
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Optimum working temperature of 37oC produces some
oligonucleotide mis-priming due to non-specific hybridization of
primers (i.e. primers anneal to wrong sequence of DNA).
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:
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Great reduction in mis-priming by oligonucleotide primers. Why? (If
you don't understand the reason, see Stringency in Section 6).
Enzyme survives high temperature so no fresh aliquots are required.
This saves time and allows easier automation of the process.
Disadvantage of Taq:
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Lack of proof-reading activity means base mis-incorporation (error)
rate is 2-4 times higher than that of 'conventional' polymerase
enzymes.
Other thermostable DNA polymerases:
Stoffel fragment
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
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Amplified fragment used directly for probing or sequencing.
Fragment length controlled by use of primers complementary to
internal sequences.
Can, therefore, produce a range of deletion mutants for sub-cloning
and analysis.
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.
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Requires degenerate pool of oligonucleotide primers of all possible
sequences.
PCR at 37oC results in primer mis-matches.
Produces amplified target DNA with variable termini.
Use of Taq polymerase and higher annealing temperature only
produces fragments with correct terminal sequences.
b) Generation of cDNA libraries
Eukaryote mRNA has poly A tail at 3' terminus.
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Small amount of cDNA can be made by reverse transcription from
only 1 or 2 mammalian cells by priming mRNA with oligo-dT.
1st strand cDNA then undergoes 3' homopolymer tailing with G
residues.
So molecules have polyT and polyG at either end.
PCR proceeds using oligo-dT and oligo-dC primers.
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
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Target DNA in clone is amplified using appropriate primers.
Amplified product then annealed with 32P labelled primer and directly
sequenced.
Avoids need for sub-cloning into sequencing vector.
problem arises when same primer used for PCR and sequencing
reactions because primers left over from PCR may compete with
labelled sequencing primers.
d) Analysis of mutations
Deletions and insertions in a gene can be detected by differences in size of
amplified product.
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Location of mutation determined by selective use of primers for
different regions of target DNA.
Or by failure to amplify i.e. when mutation lies within region
complementary to one primer.
Point mutations can be detected by using competitive nucleotide priming:
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2 or more labelled primers with single base changes used in separate
reactions.
Only perfectly matched primers yield product with high specific
activity.
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.
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Improved speed, accuracy and technical flexibility over previous
methods, e.g. pre-natal diagnosis of sickle-cell anaemia and betathalassaemia.
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Diagnosis is now possible by PCR in 1-7 days vs. 2-4 weeks by
Southern blotting, e.g. cystic fibrosis mutation can be detected within
one day using PCR.
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:
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Dot blot (spot hybridization) with oligonucleotides specific for known
mutation.
Restriction enzyme analysis (RFLP - restriction fragment length
polymorphism).
Direct sequencing.
Important to be certain of result so combination of two methods provides
confirmation.
Other conditions which can be detected with the same approach include:
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Tay-Sachs disease
phenylketonurea
cystic fibrosis (CF)
haemophilia
Huntingdon's disease
Duchenne muscular dystrophy (DMD)
In DMD, affected gene is very large - 2Mb; codes for a cytoskeletal protein,
dystrophin.
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Gene composed of coding sequences (exons) interspersed with noncoding introns of up to 35kb.
In 60% of cases, DMD arises from deletions in any of 9 specific
exons.
So multiplex (simultaneous) amplification of all 9 exons needed to
detect change.
40% of cases involve sequence polymorphisms e.g. point mutation.
Detected by RFLP analysis following PCR.
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:
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fastidious microorganisms (difficult, or impossible, to grow on artificial
growth media, e.g. Chlamydia species, Rickettsia species,
Trypanosoma species, Treponema pallidum, Pneumocystis carinii,
all viruses).
slow growing microorganisms, e.g. Mycobacterium species.
microorganisms present in small numbers in some specimens or
patients and/or at certain stages in the disease, e.g. Mycobacterium
tuberculosis, HIV.
detection of microbial genes responsible for some aspect of
pathogenesis, e.g. toxin production, antibiotic resistance, pili
formation, capsule production.
extremely hazardous microorganisms (where culture is especially
risky), e.g. Category 4 pathogens such as Ebola virus.
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.
e.g. Retroviruses
Rapid diagnosis may pre-date appearance of antibodies in blood of patient.
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However, great sensitivity required due to low numbers of viral copies
present in cells.
Viral DNA/RNA only represents a minute proportion of total cell
DNA.
Also, may only be one infected cell per 10,000 and, therefore too little
viral DNA for Southern blotting.
May need to discriminate one specific member from large family of
related viruses. So also require high degree of specificity while also
targeting conserved regions of DNA to guard against high level of
genetic variability characteristic of retroviruses.
Appropriate selection of primers can distinguish HIV1 from HIV2.
High risk of cross-contaminating sample with small amounts of
amplified DNA from previous sample requires extra precautions to
prevent false-positives.
PCR can detect 10-20 copies of viral DNA from 150,000 human cells.
Sensitivity for HIV1 and HTLV I of 80% and 100% respectively.
e.g. Mycobacterium tuberculosis
The bacterium that causes tuberculosis (TB) is conventionally identified by:
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Microscopy (e.g. acid-fast, auromine O, or fluorescent antibody
staining of the bacterial cells in the patient's sputum followed by
visualization and detection using microscopy. However, the number
of cells in the specimen is often very low (in some patients effectively
zero) meaning that false negatives are common.
Culture on solid, artificial growth media, e.g. Lowenstein-Jensen (LJ)
or Dorset's Egg. However, it can take up to 6 weeks for colonies of
Mycobacterium tuberculosis to become visible.
[Some other Mycobacterium species are even slower growers, e.g.
Mycobacterium ulcerans can take 6 months!!!]
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:
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Relatively simple to perform and therefore to standardize.
Fast- results obtainable within 24 hours.
Main legal problem with PCR is that identification is made from copied DNA
rather than original material.
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Therefore must demonstrate that errors due to mis-incorporation are
below significant level. PCR can only show the probability of a DNA
sample matching a suspect. Particularly in the past, defence lawyers
took advantage of (some?) jurors lack of knowledge of statistics
(think how many people buy National Lottery tickets and are sure
they are going to win!) to persuade them to acquit a suspect because
the DNA evidence wasn't 100% conclusive. Nowadays, most juries
will convict if the probability of the DNA specimen not originating from
the accused can be proved to be millions to one.
Another potential problem is due to cross-contamination between
samples. Unless great care is taken, the laboratory worker may
'"prove" himself, or herself, to be the murderer or rapist!
A one-way line of flow from sample preparation to PCR to DNA
typing is essential.
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:
1. A high-temperature melting step in which double-stranded target
DNA unwinds to become single-stranded.
2. A cooling step in which two sets of adjacent, complementary
oligonucleotides anneal to the single-stranded target molecules and
ligate together.
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 singlebase 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, etc.
Suggested further reading:
Brown, T.A. (2001).
Gene Cloning & DNA Analysis. (4th edition).
Blackwell.
Chapter 9 is devoted to PCR.
Primrose, S.B. et al. (2001).
Principles of Gene Manipulation.
Blackwell.
Questions 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?
END OF SECTION 8.
NOW GOT TO SECTION 9
(PROTEIN ENGINEERING).