Polymerase Chain Reaction (PCR) | Biotechnology

The below mentioned article provides a Beginner’s Guide to Polymerase Chain Reaction (PCR). After reading this article you will learn about: 1. History of the PCR 2. The Principle of Polymerase Chain Reaction 3. Requirements of PCR 4. The PCR Reaction Cycle 5. Analysis of PCR Products 6. Applications 7. Precautions and Drawbacks 8. Modifications.

Contents:

1. History of the PCR
2. The Principle of Polymerase Chain Reaction
3. Requirements of PCR
4. The PCR Reaction Cycle
5. Analysis of PCR Products
6. Applications of PCR
7. Precautions and Drawbacks
8. Modifications

1. History of the PCR:

The idea for PCR is credited to Kary-Mullis who was a research scientist in 1980s at a Cali­fornia biotechnology company called Cetus. Mullis, and five other researchers in Human Genetics Department at Cetus, demonstrated that oligonucleotide primers could be used to specifically amplify defined segments of ge­nomic DNA (or cDNA). Mullis was co-winner of 1993 Nobel Prize in Chemistry.

2. The Principle of Polymerase Chain Reaction:

Polymerase chain reaction (PCR) is a primer mediated enzymatic amplification of specifi­cally cloned or genomic DNA sequences. In this process we take the DNA with a target se­quence which we want to amplify, denature it by increasing the temperature and then use a sequence specific primer for the amplification of our target sequence by the help of a ther­mos-table DNA polymerase.

In this technique we try to reproduce an artificial environment under in vitro conditions in which the target DNA sequence undergoes multiple rounds of replication cycle to produce an enormous cop­ies of our target gene.

The process PCR has three fundamental steps:

Step 1:

Double-stranded DNA template denaturation by increasing the tempera­ture to 94 – 98°C for 30 seconds for 2 mi­nutes.

Step 2:

Annealing of two oligonucleotide primers to the single-stranded tem­plate by lowering the temperature to 50 – 65°C.

Step 3:

Enzymatic extension of primers to produce copies that can serve as tem­plates in subsequent cycles.

3. Requirements of PCR:

(a) DNA Template:

The original DNA mol­ecule that is to be copied is called the DNA template and the segment of it that will actually be amplified is known as the tar­get sequence. A trace amount of the DNA template is sufficient. This can be obtained by any one of the DNA isolation techniques discussed before.

(b) PCR Primers:

Two PCR primers are needed to initiate DNA synthesis. These are short pieces of single-stranded DNA that match the sequences at either end of the target DNA segment. PCR primers are made by chemical synthesis of DNA.

There are several computer programs available to suggest suitable primers for the process of PCR, and some of the general guide­lines are listed below:

1. Length:

Shorter primers have a tendency to go and anneal to the non-target sequence of the DNA template. This will result in production of DNA copies of having non-target sequence. The greater the complexity of the template DNA, the more likely this is to happen.

Thus, a Short primer may offer sufficient specificity when amplifying using a simple template such as a small plasmid, but a long primer may be re­quired when using eukaryotic genomic DNA as template. In practice, 20-30 nucleotides is generally satisfactory.

2. Mismatches:

Primers do not need to match the template completely, although the 3′ end of the primer should be correctly base-paired to the template, otherwise the polymerase will not be able to extend it. It is often beneficial to have C or G as the 3′ terminal nucleotide. This makes the binding of the 3′ end of the primer to the tem­plate more stable than it would be with A or T at the 3′ end.

3. Melting Temperature:

The temperatures at which the two primers can associate with the template should be rela­tively similar to ensure that they both bind at about the same time as temperatures are be­ing lowered during annealing. The similarity of melting temperatures is likely to mean that the primers have a similar nucleotide compo­sition.

4. Internal Secondary Structure:

This should be avoided in order to prevent the primer to fold back on itself and not be avail­able to bind to the template.

5. Primer-Primer Annealing:

It is also important to avoid the two primers being able to anneal to each other. Extension by DNA polymerase of two self-annealed prim­ers leads to formation of a primer dimer.

(c) Thermo-Stable DNA Polymerase:

The enzyme DNA polymerase is needed to manufacture the DNA copies. The Klenow fragment was the first DNA polymerase enzyme used in PCR. The Klenow frag­ment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin.

After enzymatic modification it retains the 5′-3′ polymerase activity and the 3′ → 5′ exonuclease activity for re­moval of pre-coding nucleotides and proof­reading, but loses its 5′ → 3′ exonuclease activity.

Klenow fragment failed to play a successful role as a polymerase enzyme for lacking a stability at high temperature. As we know that the PCR procedure involves several temperature steps, in this situa­tion we had to replenish the Klenow frag­ment during each cycle.

To solve this is­sued heat resistant DNA polymerase was required. This came originally from heat resistant bacteria living in hot springs at temperatures up to 90°C. Today Taq poly­merase from Thermusaquaticus is the most widely used PCR DNA polymerase enzyme. It is generally produced by expres­sion of the gene in E. coli.

The thermo-sta­bility of the Taq enzyme helps in its puri­fication after expression in E. coli, since- contaminating E. coli proteins can be in­activated by heating. The enzyme has 5′- 3′ DNA polymerase and 5′-3′ exonuclease activities. It will polymerize about 50-60 nucleotides per second. However, the en­zyme has a number of properties that may be disadvantageous.

1. Taq Polymerase has No Proof-Reading (3′-5′ exonuclease) Activity:

Consequently about one nucleotide in 10⁴ in­corporated is incorrect and the individual prod­ucts of PCR will be a heterogeneous popula­tion.

2. Taq Polymerase has Relatively Low Processivity:

This means that it is likely to dissociate from the template before it has synthesized a long piece of DNA.

3. Taq Polymerase is not Fully Heat Stable:

It has a half-life of about 40 min at 95°C, which means there will be significant loss of activity over the 30 or so cycles used in a typical PCR experiment. It may, therefore, be necessary to add more enzyme during the course of an ex­periment.

4. Taq Polymerase Incorporates an Ex­tra a Residue:

This is incorporated on the 3′ end of the mole­cule synthesized, and is not template encoded. A number of polymerases are available from other Thermus species. These include Tfl and Tth enzymes from Thermusflavus and Thermusthermophilus respectively.

These generally do not have 3′-5′ proof-reading ac­tivity. Polymerases are also available from other genera of bacteria (including archaebacteria), and many of these enzymes have 3′-5′ proof-reading activity (which also means, they do not usually add terminal nucleotides that are not template directed).

Proof-reading en­zymes include Tli from Thermococcuslitoraiis and Pfu from Pyrococcusfuriosus. These marine bacteria generally grow at even higher temperatures than Thermusaquaticus, and the polymerases are more ther­mo-stable than the Taq enzyme.

(d) Deoxy Nucleotide Triphosphates:

A supply of four deoxynucleotide triphos­phates, dATP, dCTP, dGTP and dTTP, are needed by the polymerase to make the new DNA.

(e) PCR Machine:

Finally we need a PCR machine to keep changing the tempera­ture. The PCR process requires cycling through sev­eral different temperatures. Because of this, PCR machines are sometimes called thermo-cyclers.

4. The PCR Reaction Cycle:

The PCR is a chain reaction because newly synthesized DNA strands will acts as template for further DNA synthesis in subsequent cycle. After 25 cycles of DNA synthesis, the products of the PCR will include, in addition to the start­ing DNA, about 10⁵ copies of the specific tar­get sequence.

PCR consists of a series of cycles of three successive reactions:

Denaturation Step:

This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes the melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

Annealing Step:

The reaction tempera­ture is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single- stranded DNA template. Typically the anneal­ing temperature is about 3-5 degrees Celsius below the T_m of the primers used.

The T_m can be determined experimentally or calculate from the following formula:

T_m = (4 x [G + C]) + (2 x [A + T])°C

Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The poly­merase binds to the primer-template hybrid and begins DNA synthesis.

Extension/Elongation Step:

The tem­perature at this step depends on the DNA poly­merase used. Taq polymerase has its opti­mum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′ to 3′ direction.

The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

For example, if one starts with a single double-stranded DNA molecule, after 20 cycles the number of mol­ecules synthesized by PCR becomes 1×10⁶, and after 30 cycles the number of the DNA mol­ecules increases to 1×10⁹.

This number can be calculated by the help of following formula:

M_f = M,x2ⁿ

where M_f is the final number of DNA mol­ecules produced by PCR, M_f is the initial amount of DNA molecules, and n is the num­ber of PCR cycles.

Final Elongation:

This single step is oc­casionally performed at a temperature of 70- 74 °C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

5. Analysis of PCR Products:

PCR is often used as a technique to gain infor­mation about the DNA template carrying a specific target sequence. Biotechnology re­search widely depends upon PCR at various situations. Hence there are several methods for analysing the products of PCR.

Following three techniques are important:

(a) Gel Electrophoresis of PCR Products:

The final results of most PCR experiments are confirmed by subjecting a portion of the amplified reaction mixture to agarose gel electrophoresis. This can inform us the validity of the PCR experiment. If the ex­pected band during gel visualization is absent, or if additional bands are present, something has gone wrong and the experi­ment must be repeated.

In some cases, agarose gel electrophoresis is used not only to determine whether a PCR experiment has worked, but also to obtain additional information. We can also determine the presence of restriction site in the template DNA by subjecting the PCR product to re­striction endonuclease prior to electro­phoresis.

This protocol is a type of restric­tion fragment length polymorphism (RFLP) analysis which has immense significance in the construction of genome maps and studying genetic diseases. Electrophoretic analysis of PCR product can also help us in identifying the insertion or de­letion mutation in the amplified region.

(b) Cloning of PCR Products:

During the cloning experiment many times we take the help of PCR directly. If the gene of in­terest is in a very less quantity, then we need to amplify it. This is done by the help of PCR which produces enough copies of our target DNA so that we can afford to start the experiment.

(c) Sequencing of PCR Products:

The se­quencing of PCR product is done by the help of an automated sequencer machine. There is no need for radio-la­belling and autoradiography. This is an im­proved way to sequence DNA because of its speed and because it can be analysed by computer rather than a person.

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6. Applications of PCR:

PCR has a number of applications especially where speed and the number of samples to be processed are important or where the amount of DNA available is very limited. Here are some of the applications.

a. DNA Sequencing:

PCR in the presence of di-deoxynucleoside triphosphates (ddNTPs), used for DNA sequencing, al­lows DNA sequencing reactions to be run successfully with very small amounts of template.

b. Diagnostic:

PCR is useful as a diagnostic tool, e.g., in the identification of specific genetic traits or for the detection of patho­gens or food contaminants. One of the first applications of PCR to genetic diagnosis was for sickle cell anaemia.

c. Forensic:

The ability to amplify DNA from regions of the genome that are highly polymorphic (and which are variable be­tween individuals) starting with samples containing very small amounts of DNA (e.g., single hairs or traces of body fluids, such as blood and semen) leads to applica­tions in forensic work.

d. Present-Day Population Genetics:

It al­lows for the determination of frequencies of particular alleles in a large collection of individuals. A particular advantage of us­ing PCR in population genetic studies is that, with appropriately designed specific primers, it may be possible to amplify DNA from one organism that cannot be sepa­rated from others, such as a particular bacterial strain in a mixed population.

(Such primers will anneal to the target DNA from the organism of interest, but not to DNA from others.)

e. Archaeology and Evolution:

PCR can be used with old material as well as more re­cent samples, and it is often possible to amplify ancient DNA from museum speci­mens and archaeological remains. Mostly mitochondrial DNA or chloroplast DNA is used. This allows inferences to be made about the origins of particular populations or species.

7. Precautions and Drawbacks:

i. Size:

The size of fragments that can be am­plified is limited by the processivity of the polymerase used. Using a mix­ture of polymerases that includes a proof­reading enzyme increases the size of pro­duct that can be obtained (up to 10 kbp or more), because incorrectly incorporated nucleotides can be removed rather than causing chain termination.

ii. Amplifying the Wrong Sequence:

PCR depends on the ability of the primers to anneal to the correct sequence, and this depends on the conditions of annealing (ionic concentration, temperature, etc.) and the actual sequence (or sequences if mixed sites are included) of the primers.

It is possible for primers to anneal to the “wrong” part of the target DNA, through chance complementarity. If this happens and the primers anneal in the correct ori­entation to each other (i.e., directing syn­thesis towards each other) and at sites that are not too far apart, then the result is the amplification of a sequence other than the desired one.

The possibility of incorrect annealing may be avoided by use of longer primers, which will be more specific in their annealing sites. Raising the temperature and adjust­ing the concentration of magnesium ions (which stabilize primer-template binding) can be used to increase the specificity of primer binding.

iii. Contamination:

Because of the extraor­dinary sensitivity of PCR, there is a par­ticular danger of contaminating the DNA sample to be amplified with extraneous material. This is particularly important when using material containing only small amounts of DNA, as with archaeological work.

Contamination might be of labora­tory origin (e.g., from aerosols created by pipetting solutions containing related DNA sequences, including material amplified previously by PCR) or of external ori­gin (perhaps by bacterial, fungal or human contamination of sample tissue).

Laboratory contamination can be mini­mized by precautions such as careful use and design of pipettes, separation of the pre-PCR and post-PCR stages of an experi­ment into different rooms. Contamination from other sources can be reduced by care­ful handling and preparation of a sample before amplification.

iv. Sequence Heterogeneity:

Amplification may give rise to a mixture of molecules of slightly different sequences.

A mixture could arise for several reasons:

(a) Heterozygosity:

If the template DNA came from an individual heterozygous at the locus in question, each of the alleles present should be represented in similar quantities in the PCR prod­ucts.

(b) Population Heterogeneity:

If the tem­plate DNA came from several individu­als rather than a single one, heteroge­neity in the population may give rise to heterogeneity in the products.

(c) DNA Damage and Polymerase Error:

Heterogeneity can also arise from damage to DNA before amplification, especially if the sample has not been carefully preserved. Therefore, this is particularly likely to be a problem with archaeological and forensic material.

v. Jumping PCR:

When degraded DNA is amplified, it may be that any given sample molecule is not long enough to span the entire distance between the two priming sites. The result in the first round of syn­thesis would be extension of the primer to the end of a fragmented molecule, but not all the way to the second primer site.

How­ever, on a subsequent round of synthesis, the truncated amplification product may anneal to a different DNA fragment that contains the remaining region intact. This would then allow synthesis of the full PCR product. This is called jumping PCR. So it is sometimes possible to generate PCR products that are longer than any indi­vidual template molecule. This can be ad­vantageous when amplifying badly de­graded DNA.

8. Modifications:

a. Hot-Start PCR:

As soon as the PCR re­agents have all been mixed together, it is possible for the DNA polymerase to start synthesis. This may happen while the re­action mixture is being heated for the first time, and is at a temperature low enough to allow non-specific annealing of primer to template, generating a range of non-spe­cific products.

This problem would be pre­vented if DNA synthesis could not take place until the first cycle had reached its maximum temperature. This is the basis of hot-start PCR. In the simplest form, the DNA polymerase is not added to the reaction tubes until they have reached the DNA melting temperature of the first cycle. This is satisfactory where small numbers of samples are being processed, but not with large numbers.

b. Touch-Down PCR:

The annealing tem­perature used in conventional PCR is usu­ally several degrees below the maximum at which primers can remain bound to tem­plate, to ensure stable binding. However, this use of a lower temperature permits a small amount of mismatching between primers and template, which may allow primers to bind to incorrect sites and gen­erate spurious products.

The effects of this can be reduced with touch-down PCR. In this, a high annealing temperature is used initially (at which even correct bind­ing may not be possible). The annealing temperature is reduced in subsequent rounds. There will, therefore, come a point at which correctly matched primer-tem- plate annealing is just possible, but incor­rect matching is not and the desired prod­ucts will be the most abundant.

c. Nested PCR:

Here, two successive PCRs are carried out. The first PCR uses a con­ventional template. The products of the first PCR are then used as the template for the second PCR, with primers that are designed to anneal within the desired prod­uct of the first PCR.

Although the first PCR may generate some non-specific products in addition to the desired products, it is unlikely that the non-specific products will also contain annealing sites for both the primers used in the second PCR. Thus, only the desired products from the first PCR are likely to be suitable templates for the second.

d. Inverse PCR:

It is possible to arrange for the amplification of sequences outside the primers, in a technique called inverse PCR (IPCR). In this technique the sample DNA is first cut with an enzyme outside the region whose sequence is already known.

The resulting linear molecules are then circularized, by ligation under condi­tions that favour intermolecular reactions. A second restriction digestion is then done, using an enzyme cutting within the region of known sequence.

The result is now that the first fragment containing this sequence has been turned ‘inside out’, leaving known sequence on the outside and the material that had previously been flanking it within. Primers complementary to the known sequence on the outside of the mole­cule can now be used to amplify the region of interest between them.

e. Reverse Transcriptase PCR:

It is often convenient to amplify RNA molecules, per­haps as a precursor to cloning them, or to estimate the abundance of a particular mRNA in a sample. This is usually done by having a round of reverse transcription, using a reverse transcriptase enzyme and a single primer, to make a single strand of cDNA prior to the PCR itself.

The primer for reverse transcription could be oligo-dT for general cDNA synthesis from polyadenylated messages, or it could be specific to a particular message.

f. In Situ PCR:

It is possible to carry out PCR using permeabilized tissue, such as thin sections on a microscope slide. This requires a specially adapted PCR machine to accommodate the slide. If the PCR prod­uct can be detected (perhaps by hybridiza­tion, also in situ), then this allows one to identify where in the tissue the target nucleic acid is located.

g. Asymmetric PCR:

By reducing the amount of one of the two primers, it is pos­sible to arrange for preferential amplifica­tion of one of the strands, resulting in a preparation of single-stranded DNA, which has a number of uses in molecular biology. Preferential amplification of one strand in this way is known as asymmet­ric PCR.

h. Anchored PCR:

Anchored PCR is applied when only one piece of sequence (and therefore, one priming site) for the region of interest is known. The aim is to attach the region to be amplified to a piece of known sequence and then to use that as the second priming site.

There are two ways in which this can most easily be done. One is to fragment the sample DNA and ligate it to molecules of known sequence, such as a vector. This known sequence is used as the basis for designing one of the two PCR primers. The second method is to add tails enzymatically to the sample DNA or the molecules produced after the first round of synthesis.

i. Emulsion PCR:

In a conventional PCR, the reactions are carried out inside plastic tubes. It is possible to incorporate all the reagents inside lipid droplets and carry out PCR on a much smaller scale. This has certain advantages. It is possible to in­crease and decrease the temperature of small droplets very quickly.

In addition, if each droplet contains a single template molecule at the start, then all the products in an individual droplet result from the am­plification of a single template molecule. The method is also called droplet PCR.

j. Isothermal Amplification:

The repeated heating and cooling required by PCR lim­its how quickly the process can be carried out. Loop-mediated isothermal ampli­fication (LAMP) has been developed, which allows templates to be amplified at a constant temperature (typically around 65°C).

It uses a DNA polymerase with strand-displacing activity and avoids the need for heating to high temperatures. This method is used for the detection of pathogens outside of specialist laboratories.

k. Real Time PCR:

It is possible to use PCR to estimate the abundance of a particular nucleic acid molecule in a sample. This can be done by real time PCR. This can be done in two ways.

In the first, a fluorescent, double-stranded DNA (dsDNA)-binding dye (such as SYBR green) is present in the PCR. As dsDNA prod­uct accumulates, the amount of fluorescence from the dye increases, and this can be de­tected.

The experiment requires a PCR ma­chine that is also equipped with a fluorescence measurement facility. Because the method simply detects dsDNA, it measures the amount of PCR product at a given time regardless of whether it is from the correct region.

The second approach to real-time PCR al­lows detection of a specific product, rather than dsDNA in general, and uses a specially syn­thesized probe oligonucleotide. This probe is designed to anneal within the region to be amplified and carries a fluorescent reporter dye at one end and a quencher at the other end of the molecule.

If the quencher and the reporter are in close proximity (i.e., attached to the same oligonucleotide), then the quencher stops the reporter from fluorescing.

During PCR, the probe will anneal to single-stranded DNA within the target region. When the poly­merase meets the annealed probe, the 5′-3′ exonuclease activity of the enzyme degrades the probe, liberating the reporter from the quencher. Thus, the fluorescent reporter ac­cumulates during the course of the PCR. This type of PCR mechanism is shown in the dia­gram given above.