POLYMERASE CHAIN REACTION

POLYMERASE CHAIN REACTION

 The polymerase chain reaction is very different from gene cloning. Rather than a series of manipulations involving living cells, PCR is carried out in a single test tube simply by mixing DNA with a set of reagents.
The basic steps in a PCR experiment are as follows-
(1) The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature.
(2) The mixture is cooled down to 50-60°C. The two strands of each molecule could join back together at this temperature, but most do not because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions.
 The temperature at which annealing of the primers to the template DNA occurs depends upon the length and sequence of the primer, and the level of specificity required in a particular PCR reaction.
(3) The temperature is raised to 74°C. This is the optimum working temperature for the Taq DNA polymerase that is present in the mixture. All we need to understand at this stage is that the Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules, during this step of the PCR. Now we have four strands of DNA instead of the two that there were to start with.
(4) The temperature is increased back to 94°C. The double-stranded DNA molecules, each of which consists of one strand of the original molecule and one new strand of DNA, denature into single strands. This begins a second cycle of denaturation-annealing-synthesis, at the end of which there are eight DNA strands. By repeating the cycle 25 times the double-stranded molecule that we began with is converted into over 50 million new double-stranded molecules, each one a copy of the region of the starting molecule delineated by the annealing sites of the two primers.



Figure 4.1. The steps of a PCR experiment. The two DNA strands of the target DNA molecule, shown in red and blue to differentiate them, are denatured, or separated, by heating. The boxed regions depict unique sequences within the target DNA to which the oligonucleotide primers will bind. Once the strands are separated, they are then cooled in the presence of oligonucleotides that are complementary to each strand. This results in the annealing of the oligonucleotides to their complementary DNA sequence. The oligonucleotides are designed such that their 3_-ends face each other. The oligonucleotides are then extended using DNA polymerase in the presence of the four deoxynucleotide triphosphates. This cycle of denaturing, annealing and extension is repeated 20–30 times to result in a massive amplification of the DNA in between the two oligonucleotide binding sites.

 There is a limit to the length of DNA sequence that can be copied by PCR. Five kilobases (kb) can be copied fairly easily, and segments up to 40kb can be dealt with using specialized techniques, but this is shorter than the lengths of many genes, especially those of humans and other vertebrates.

What is not so obvious, however, is how DNA synthesis is terminated. If we inspect the results of a typical specific PCR reaction on an agarose gel (Figure 4.2), we see that a single, discrete band is formed. This suggests that the DNA fragments produced are homogenous and that they begin and end at the same point.
During the first PCR cycle, the two target DNA strands are separated and DNA replication initiates at the point of primer binding. The two newly synthesized DNA strands at the end of cycle 1 will each have defined 5’-ends, as dictated by the site of primer binding, but ill defined
3 ‘-ends.

DNA synthesis will not terminate at a specific point, but will only stop during the heat denaturation step of cycle 2. Each of the DNA strands present at the end of cycle 1 will proceed into the next cycle of PCR, and each will act as a template for the binding of new primers. In cycle 2, primer binding to both the original template strands and the strands synthesized during cycle 1 will occur. Primer binding to the original template strands will result in the formation of the same products that were made during cycle 1. However, primer binding to the DNA strands produced in cycle 1, followed by replication, will result in the formation of a DNA strand with both a defined 5’-end and a defined 3’-end. This occurs because DNA replication will terminate when there is no more DNA sequence to copy. Thus, at the end of cycle 2, two DNA strands are formed (shown in purple) that have a defined 5’- and a defined 3’-end.
These are, however, base-paired to DNA fragments that have ill defined 3’-ends. Again, the products from cycle 2 of the PCR process will go forward into cycle 3 and, again, each DNA strand can be used as a template for primer binding. At the end of cycle 3, two double-stranded DNA molecules are formed that have 5’- and 3’-ends beginning and ending at the positions of primer binding within the original target DNA sequence. These are the boxed sequences shown in Figure.
After 25 cycles, typical for many PCR experiments, an amplification of about 30 million-fold is expected, and amplifications of this order are actually attained in practice. All PCR reactions are ‘contaminated’ with small quantities of DNA fragments that have incorrectly formed ends, but the massive amplification of specific DNA fragments means that these are almost insignificant.
PCR Reaction Conditions
A typical PCR experiment will contain the following components:
• DNA (0.01–0.1 μg)
• Primer 1 (20 pmol)
• Primer 2 (20 pmol)
• Tris-HCl (20 mM, pH 8.0)
• MgCl2 (2 mM)
• KCl (25 mM) or KCl (10 mM) and (NH4)2SO4 (10 mM)
• Deoxynucleotide triphosphates (50 μM each of dATP, dCTP, dGTP, dTTP)
• Thermostable DNA polymerase (2 units)
• A total reaction volume of 50–100 μL.
 Of the reagent components of the reaction (Tris, KCl and MgCl2), the concentration of magnesium ions in the reaction plays a significant role in the success of a PCR reaction.
 Magnesium is required for the DNA polymerase to function, but the specificity of any particular PCR reaction is dependent upon the concentration of magnesium used. At low concentrations of magnesium, the reaction fails because the polymerase is insufficiently active. At high concentrations of magnesium, the reaction loses specificity and multiple products are produced.
 The optimum magnesium concentration needs to be determined empirically for each separate PCR primer set, but will usually be in the range of 1–5 mM.
 The buffer and salt components of the reaction (Tris and KCl) are usually held constant, although some protocols reduce the level of KCl to encourage DNA polymerase to remain on the template for longer and achieve a greater length of amplified product.
 Once the PCR reaction has been set up, it is often covered with a layer of mineral oil to prevent evaporation of the sample during heating – alternatively, a PCR machine with a heated lid will prevent evaporation.
Typical cycling conditions for a PCR experiment might be
• 94 ◦C, 30 s – denaturation
• 60 ◦C, 30 s – annealing
• 72 ◦C, 1 min – extension.
 Early protocols included an initial denaturing step (94 ◦C, 2 min) to ensure that the initial template DNA was fully single stranded. This is usually not included now since long exposures to high temperature will induce nicks in the template DNA.
 The number of PCR cycles that are performed during an individual experiment depends upon both the amount of initial DNA template in the reaction and the amount of DNA required after the amplification process.

THERMOSTABLE DNA POLYMERASES
 The bacterium Thermus aquaticus was first discovered in several hot springs in the Yellowstone National Park, The organism has a temperature tolerance range between about 50 and 80 ◦C, and its optimum growth temperature is around 70 ◦C.
 Taq DNA polymerase is a monomeric enzyme.
 The enzyme itself is thermo stable; it replicates DNA at 74 ◦C and remains functional even after incubation at 95 ◦C.
 The enzyme includes a 5’ to 3’ polymerase activity and a 5’ to 3’ exonuclease activity, but it lacks a 3’ to 5’ exonuclease (proofreading) activity. The lack of proofreading activity means that if an incorrect base is inserted into the extending polynucleotide chain, it cannot be removed and consequently Taq DNA polymerase is error prone and will introduce mutations into amplified PCR products.
 This level of error introduction does not, however, affect the affect the outcome of a PCR experiment. If the PCR is being performed merely to identify the presence or absence of a gene within a particular target DNA molecule
 However if the amplified gene is to be studied functionally, then PCR errors may significantly affect the experiment. The problem of error introduction does mean, however, that PCR products should be subjected to DNA sequence analysis before they are used in cloning experiments.
 Another functional aspect of Taq DNA polymerase that impinges upon them sequence of the final PCR product is the tendency of the enzyme to incorporate a deoxynucleotide (often an adenosine) in a template-independent manner on the 3’-end of the newly synthesized DNA strand. A consequence of this activity is that PCR products produced by Taq do not have blunts ends, but have a single 3’ A residue overhang. This property has been exploited to aid the cloning of PCR products.
Some of the other thermo stable DNA polymerases, e.g. Pfu polymerase isolated from the organism Pyrococcus furiosis, do possess a 3’ to 5’ exonuclease proofreading activity, and so their mutation rate is reduced. It also produced blunt ends like T. litoralis ,T.gorgonarius while T. aquaticus & T. flavus produced 3’ A ends.

 The 5’ to 3’ exonuclease activity of Taq DNA polymerase means that the enzyme is able to degrade the oligonucleotide primers within the PCR reaction.
 This is particularly relevant during the first denaturing step of cycle 1, when the oligonucleotides are not bound to the DNA template, and the polymerase is free in solution. During the first heating cycle, the temperature of the PCR mix rises from room temperature (or 4 ◦C if the reaction was set up on ice) to 94 ◦C. This means that, at some point, the temperature within the tube will be 72 ◦C – the optimum for the polymerase – but the enzyme will be unable to replicate DNA since none of the oligonucleotides are bound to the template DNA. Passing through the temperature of the enzyme without replication occurring will tend to result in primer degradation, and subsequent inefficient PCR. To overcome this problem, and to prevent non-specific PCR products being synthesized prior to cycling, Taq DNA polymerase can be added to the reaction mix already at 94 ◦C. This ‘hot start’ increases both the yield and specificity of the PCR reaction.
 Alternatively, Taq DNA polymerase can be mixed with a specific antibody that binds to the enzyme and inhibits its activity. The antibody – enzyme complex inhibits replication at low temperatures, but the complex irreversibly dissociates at high temperature, after which the enzyme is unhindered in its function.
 A mixture of the two polymerases (15 parts Taq and 1 part Pfu) has been found to efficiently amplify DNA fragments up to 35 kbp in length with high fidelity.

TEMPLATE DNA
 Almost any DNA sample can be used as a template for a PCR reaction, including linear, closed-circular and supercoiled plasmid DNA, genomic DNA, cDNA etc. The source of the DNA is immaterial, since PCR is merely a sequence directed event.
 The only requirement is that the primer binding sites, and the sequence between them, are intact.

Q.1 How many copies of the target sequence does this amount of DNA correspond to? If you add 1 μg of human genomic DNA to a PCR reaction,
[1 μg of human genomic=10-6 gm of human genome]
This is equivalent to ---------------------
[1 ×10-6 / (6.4 × 109× 650) = 2.4 × 10-19mol, ]
Since human DNA contains Approximately 6.4 × 109 bp of DNA and the average molecular weight of a base pair is 650 Da.
(no’ of moles=wt/Mw)
Therefore 1 μg of human DNA corresponds to 2.4 × 10-19mol × 6 × 1023 (Avogadro’s number) = approximately 144 000 molecules.
That is, a single gene will be represented 288 000 times in 1 μg of genomic diploid DNA.
OLIGONUCLEOTIDE PRIMERS;
• The success, or otherwise, of a PCR experiment is almost wholly dependent upon the oligonucleotide primers. The primers need to be designed such that one recognizes the sense strand of the DNA to be replicated (i.e. is the same sequence as the antisense strand) while the other recognizes the antisense strand of the target DNA (i.e. is the same sequence as the sense strand).
Primers will have the following characteristics.
• They will be between 17 and 30 nucleotides in length – sufficient to allow unique annealing to a single sequence within a genome.
• They will have a GC content of approximately 50 per cent.
• The annealing temperatures of the pair of primers –
Calculated from the equation 2(AT) + 4(GC) – used in a single experiment should be approximately equal.
• Sequences with long runs of a single nucleotide should be avoided to prevent binding of the primer to repetitive sequences in the target DNA.
• Individual primers should not contain sequences that are complementary. For example, a primer of the sequence 5’-GAGATCGATGCATCGATCTC- 3’ may appear a good choice for a PCR primer (20 nucleotides long, 50 per cent GC content and not containing repetitive sequences), but it is palindromic and will form a hair-pin structure if the 5’-end binds to the 3’-end. This secondary structure is undesirable, and will effectively remove the primer from the PCR reaction so amplification of the target will not occur.
• There should be no complementarity between the two primers or the 3’ends of a single primer. For example, the following two primers again appear to be good choices: 5’-GATCGATCGATACGTGATCC-3’ and 5’- CGTAGCTAGCTAGGATCACG-3’. However, the 3’-ends of the primers are complementary to each other and primer dimers can form, which will be replicated during the first cycles of the PCR reaction:


SYNTHESIS OF OLIGONUCLEOTIDE PRIMERS
• The building blocks used for synthesis are DNA phosphoramidite nucleosides (sometimes called monomers).
• These are modified to prevent branching or other undesirable side reactions from occurring during synthesis.
• They are modified at the 5’-end (with a dimethoxytrityl group) and at the 3’-end (with a β-cyanoethyl protected 3’-phosphite group), and may also include additional modifiers to protect reactive primary amines in the nucleoside ring structure.

The phosphoramidite approach to oligonucleotide synthesis proceeds in four
Steps—
• The polystyrene is loaded into a small column that serves as the reaction chamber. Bases are added to the growing chain in a 3’ to 5’ direction (opposite to enzymatic synthesis by DNA polymerases).
• Synthesis is begun using polystyrene that is already derivatized with the first base, which is attached via an ester linkage at the 3’-hydroxyl. Primer synthesis initiates with cleavage of the 5’ trityl group by brief treatment with acid. Monomer activated by tetrazole is coupled to the available 5’-hydroxyl (Figure 4.6(c)) and the resulting phosphite linkage is oxidized to phosphate by treatment with iodine (Figure 4.6(d)). This completes one ‘cycle’ of oligonucleotide synthesis.

• The nucleoside condensation reaction is highly efficient, with less than 1 per cent of the 5’-hydroxyl groups not reacting with the incoming nucleoside. To prevent these unreacted molecules participating in subsequent reactions, and resulting in unwanted truncation deletions, the unreacted 5’-OH groups are blocked by acetylation (capping) with acetic anhydride before the oxidation step.
• After synthesis is complete, the oligonucleotide is cleaved from the solid support with concentrated ammonium hydroxide at room temperature. Continued incubation in ammonia at elevated temperature will deprotect the phosphorus via β-elimination of the cyanoethyl group and also removes the protecting groups from the heterocyclic bases. The finished oligonucleotide can be purified from contaminating chemicals by precipitation, and the full-length sequence is usually isolated by HPLC purification.

PRIMER MISMATCHES
• The oligonucleotide primers that are used in a PCR experiments need not match the target sequence exactly. This is particularly relevant when trying to make mutations, or deliberate changes, in the amplified DNA sequence or when attempting to search for gene sequences that are homologous to one already known.
• The only place within the primer sequence that must match the target sequence exactly is the extreme 3’-end of the primer. If the 3’-end of the primer does not precisely match the target sequence, then the polymerase will not efficiently extend the primer. A consequence of this is that the PCR will be inefficient, or will fail completely. This property has, however, been exploited in the diagnosis of point mutations within genes.
 Consequently, any base changes between the primer and the template DNA will be carried forward into the amplified product. Since we cannot introduce mutations at the 3’-end of the primer, a favourite location to introduce changes is the 5’-end of the primer.
• In this case, we are amplifying the same sequence as shown in Figure 4.5.
• However, this time the oligonucleotide primers contain additional sequences at their 5’-ends. In the case of primer 1, it contains the recognition sequence for the EcoRI restriction enzyme at the 5’-end of the sequence used to recognize the GAL4 gene. This primer and the primer shown in Figure 4.5 will bind to the same template DNA sequence with approximately the same affinity. The EcoRI recognition sequence does not match the template sequence exactly, but the mismatches are not sufficient to prevent specific primer binding. The EcoRI recognition sequence will, however, be incorporated into the final PCR product as shown in Figure 4.7. Primer 2 in this figure contains the recognition site for the BamHI restriction enzyme, which will also be incorporated into the final product.

• Cloning of the final product now becomes straightforward after cutting with the two restriction enzymes – having first ensured that there are no EcoRI or BamHI restriction enzyme sites in the PCR product itself!
• Often restriction enzymes require more DNA than just their recognition site in order to cleave efficiently. Therefore three to six additional residues are usually added to the 5’-end of the primer before the restriction enzyme recognition site. These are often G or C (termed a GC clamp) to provide the maximum level of annealing between the two DNA stands and efficient cleavage by restriction enzymes.
• The second major use of mismatched primers is in the search for genes encoding a particular protein, and in the search for homologous genes.
PCR IN THE DIAGNOSIS OF GENETIC DISEASE
The Waardenburg syndrome are caused by mutations in the PAX-3 gene
CLONING PCR PRODUCTS
• We have already seen, it is possible to clone PCR products by the insertion of extra sequences on the 5’-ends of primers such that restriction enzyme recognition sites are incorporated.
• It is also possible to clone PCR products directly, taking advantage of the terminal tranferase activity of Taq DNA polymerase to add a template-independent A residue to the 3’-end of PCR products.
• A consequence of this activity is that most of the DNA molecules amplified using Taq polymerase possess single 3’-A overhangs. In a process termed TA cloning, these can be ligated to a linearized ‘T-vector’, which has single 3’-T overhangs on both ends to allow direct, high-efficiency cloning of PCR products The complementarity between the PCR product 3’-A overhangs and vector 3’-T overhangs aids efficient ligation that does not occur with blunt-ended DNA molecules.


RT–PCR
Reverse transcription – polymerase chain reaction (RT–PCR) has been devised as a method of RNA amplification and quantitation after its conversion to DNA.
RT–PCR can be used for cloning, cDNA library construction and probe synthesis.
The technique consists of two parts ------
1. The synthesis of DNA from RNA by reverse transcription (RT)
2. The subsequent amplification of a specific DNA molecule by polymerase chain reaction (PCR).
The RT reaction uses an RNA template (typically either total RNA or polyA+ RNA), a primer (random or oligo dT primers), dNTPs, buffer and a reverse transcriptase enzyme to generate a single-stranded DNA molecule complementary to the RNA (cDNA).
The cDNA then serves as a template in the RT-PCR reaction. During the first cycle of PCR, the single DNA strand is made double stranded through the binding of another, complementary, primer and the action of Taq DNA polymerase.


REAL-TIME PCR
Real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, whose signal increases in direct proportion to the amount of PCR product in a reaction.
In the simplest form, the reporter is the double-strand DNA-specific dye SYBR Green. SYBR Green binds double-stranded DNA, probably in the minor groove, and, upon excitation, emits light.
Thus, if the dye is included in a PCR reaction, as a PCR product accumulates the fluorescence increases. The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive.
The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer dimers and other non-specific reaction products, which can

 The alternative method for quantifying PCR products is TaqMan, which relies on fluorescence resonance energy transfer (FRET) of hybridization probes for quantitation.
 TaqMan probes are oligonucleotides that contain a fluorescent reporter dye, typically attached to the 5’ base, and a quenching dye, typically attached to the 3’ base.
 Fluorescence increases in each PCR cycle, proportional to the rate of probe cleavage, and is measured in a modified thermocycler.