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Polymerase Chain Reaction

1. Introduction

The polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

2. History

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (122 to 176 °F)) environments paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: “Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat.” He was awarded the Nobel Prize in Chemistry in 1993 for his invention, seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis’ work, and whether he had been the sole inventor of the PCR principle.

3. PCR principles and procedure

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

DNA template that contains the DNA region (target) to be amplified.

Two primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target.

Taq polymerase or another DNA polymerase with a temperature optimum at around 72 °C.

Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.

Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis

Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

3.1 Procedure

The PCR usually consists of a series of 20-40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.

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 DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

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 °C below the Tm of the primers used. 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, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand. 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.

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.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.

3.2 PCR stages

The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.

Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

Plateau: No more products accumulate due to exhaustion of reagents and enzyme.

3.3 PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

4. Variations on the basic PCR technique

Allele-specific PCR: a diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3′ ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. See SNP genotyping for more information.

Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.

Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

Hot-start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase’s activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.

Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.

Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.

Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers (“smalligos”) as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.

Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR.

Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3′ of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Overlap-extension PCR: a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.

Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5′ end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).

Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), ‘Bridge PCR’ (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR  (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal ’step’ to favour solid support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.

Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.

PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.

Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific ‘two-sided’ PCR than conventional ‘one-sided’ approaches (using only one gene-specific primer and one general primer – which can lead to artefactual ‘noise’) by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5′RACE LaNe and 3′RACE LaNe.

5. Application of PCR

Selective DNA isolation: PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR ‘fingerprints’ methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.

Amplification and quantification of DNA: Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

PCR in diagnosis of diseases: PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus (“viral load”) in a patient can also be quantified by PCR-based DNA quantitation techniques.

6. Patent wars

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by Du Pont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on March 28, 2005.

7. PCR Components

Choice of Polymerases for PCR: One of the important advances which allowed development of PCR was the availability of thermostable polymerases. This allowed initially added enzyme to survive temperature cycles approaching 100 °C.

Buffers and MgCl2 in PCR reactions: A typical reaction buffer for PCR contains:

10 mM Tris, pH 8.3

50 mM KCl

1.5 mM MgCl2

0.01% gelatin

The MgCl2 concentration in the final reaction mixture is usually between 0.5 to 5.0 mM, and the optimum concentration is determined empirically (typically between 1.0 – 1.5 mM). Mg2+ ions:

form a soluble complex with dNTP’s which is essential for dNTP incorporation

stimulate polymerase activity, increases the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction.

Generally, low Mg2+ leads to low yields (or no yield) and high Mg2+ leads to accumulation of nonspecific products (mispriming).

Primers: Primers are needed for initializing the polymerase activity. The DNA polymerase needs a short stretch of nucleotide sequence to bind to and add nucleotides in the 5′ to 3′ direction. Primers are designed as per the need of the experiment. The common rules for designing PCR primers are as follows:

1. To have specificity of amplification, you must have a unique sequence for not only the template you are using but also any DNA or cDNA in your template preparation. Often you are amplifying from DNA prepared from tissues, cells in culture, or bacterial preparations. Remember it does not take much DNA to generate a product from PCR amplification. FindPatterns is a good program to run against the part of a database which would be relevant for your DNA preparation, to find out if there are other DNAs to which your primer will anneal.

2. The 3′ end of the primer should be G or C (S in the IUPAC-IUB notation) in order to increase correct annealing at the site of addition of bases. G-C base pairs have stronger hydrogen bonding.

3. Base pairing with itself decreases the ability of a primer to correctly anneal to the template. In general don’t use primers which have more than 4 bases in a stem. You can use StemLoop to check for this.

4. Primer pairs which can anneal to each other will. If the base pairing includes one or both 3′ ends, the polymerase will simply extend the end of the primer against the other primer as template resulting in the formation of a primer dimer:

Primer dimer formation uses up all the polymerase in the reaction and results in a lack of other product.

5. If the G+C% of two primers are very different they will have different Tms and will not anneal at the same temperature. If all the nucleotides at the 5′ end of the primer are G or C and all at the 3′ end are A or T, although the overall ratio is fine, the 3′ end will not anneal at the predicted temperature.

6. Primer length should be long enough to insure that the sequence will be unique for the template and not so long as to affect the Tm and annealing too radically. But successful primers have been as small as 14 nucleotides and much longer than 30 nucleotides.

7. Approximate Tm can be calculated manually using the formula:

Tm = 2AT + 4GC

Much better calculations use the nearest neighbor algorithms discussed in the Prime program.

8. Maximum yields are generated with Taq polymerase when the length of the product is 100-600 base pairs. Different polymerases have different degrees of processivity and will have different optimal sizes.

Calculations needed for PCR:

Calculating the melting temperature (Tm) of primers:

The Tm of primer hybridization can be calculated using various formulas. The most commonly used formula is:

(1) Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]

This formula was determined originally from oligonucleotide hybridization assays, which were performed in 1 M NaCl, and appears to be accurate in lower salt conditions only for primers less than about 20 nucleotides in length.

The common wisdom is that the Tm is more like 3-5 °C lower than the value calculated from this formula.

(2) Tms = 81.5 + 16.6(log10[J+]) + 0.41(%G+C) – (600/l)

Where [J+] = the molar concentration of monovalent cations (e.g. Na+ from NaCl), and l = the length of oligonucleotide. (%G+C) is the actual percentage value and not a fractional representation (i.e. the value to insert for a primer which had 90 % G+C content would be “90″ and not “0.90″).

This formula is reportedly useful for primers of 14 to 70 bases in length.

(3) Tmp = 22 + 1.46([2 x (G+C)] + (A+T))

This formula is reportedly useful for primers of 20-35 bases in length.

The calculated annealing temperature is only a reference temperature from which to initiate experiments.

The actual annealing temperature may be 3-12 °C higher than the calculated Tm.

The actual annealing temperature condition should be determined empirically.

The highest annealing temperature which gives the best PCR product should be used.

Calculating primer concentrations:

The molar concentration of a primer can be calculated based on the absorbance of the primer at 260 nm (A260) and the molar extinction coefficient for the primer at this wavelength.

The molar extinction coefficient for the primer can be calculated by knowing the sequence of the primer and then summing the molar extinction values for the individual bases which comprise the primer.

The individual bases have the following molar extinction coefficients at 260 nm:

A 1.0 molar solution of dT has a value of 8,400 absorbance units at 260 nm.

A 1.0 molar solution of dA has a value of 15,200 absorbance units at 260 nm.

A 1.0 molar solution of dG has a value of 12,010 absorbance units at 260 nm.

A 1.0 molar solution of dC has a value of 7050 absorbance units at 260 nm.

For example, the primer 5′ TAGC 3′ would have a molar extinction coefficient of 42,660 at 260 nm. Likewise, a 10 micromolar solution of this primer would give an absorbence of 0.427 at 260 nm.

About the Author

Author has ~6 years of research experience in molecular diagnostics and transgenic development. Currently he is working as Application Scientist in Spinco Biotech Pvt. Ltd.

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Nady Powered Floor Wedge Monitor – 12 Woofer, 125-Watts $359.64 Frequency response: 60Hz-18kHz. Power and clipping LEDs. Durable black carpet cover, plastic protective corners and recessed carry handles….

Bazooka BT1224DVC BT Series 12-Inch 4-Ohm Dual Passive Tube $249.95 Bazooka Bass TubesBazooka Bass Tubes enclosures are optimized for corner loading. The Bass Tubes enclosure’s port and woofer are on the same plane and in close proximity for maximum corner-loading results. And, just like a megaphone amplifies a cheerleader’s voice, the corner of your car amplifies the concentrated bass output of the Bass Tubes enclosure. The Bass Tubes enclosure’s advantage is it …

Bazooka MBTA10100 10-Inch 100-Watt Marine Subwoofer $399.95 As all in one solutions go, you simply can?t beat the A100 Amplified Bass Tubes by Bazooka. Designed to be a simple-to-install, yet effective upgrade to your boats audio system, the A100 Bass Tubes feature a 100 watt, 2-channel amplifier built right in, coupled to a dual voice coil woofer. Since the amplifier has a built in crossover to remove the high frequencies the woofer cannot play from the s…

4 X 10 Horn Tweeter $17.07 4″ X 10″ Horn Tweeterli200 watts maximum power liFrequency response: 1kHz – 15kHzli20 oz. magnetli8 ohms impedance…

Pyle Home PCA1 30-Watt Stereo Mini Power Amplifier $82.99 Compatible with Walkman and MP3 via 3.5mm to RCA converter2 x 15 watts 10% RMS at 4 ohmsPower on LED indicator and master volume controlRCA cable L/R input4 push type speaker L/R terminals…

Tube Guitar Amplifier Service and Overhaul - $59.99 Tube Guitar Amplifier Service and Overhaul -

Amplifier $140.19 Generally, an amplifier or simply amp, is any device that changes, usually increases, the amplitude of a signal. The relationship of the input to the output of an amplifierusually expressed as a function of the input frequencyis called the transfer function of the amplifier, and the magnitude of the transfer function is termed the gain. In popular use, the term usually describes an electronic amplifier, in which the input signal is usually voltage or current. In audio applications, amplifiers operate loudspeakers used in PA systems to make the human voice louder or play recorded music. Amplifiers may be classified according to the input (source) they are designed to amplify (such as a guitar amplifier, to perform with an electric guitar), the device they are intended to drive (such as a headphone amplifier), the frequency range of the signals (Audio, IF, RF, and VHF amplifiers, for example), whether they invert the signal (inverting amplifiers and noninverting amplifiers), or the type of device used in the amplification (valve or tube amplifiers, FET amplifiers, etc.). Author: Miller, Frederic P./ Vandome, Agnes F./ McBrewster, John Binding Type: Paperback Number of Pages: 220 Publication Date: 2009/11/25 Language: English Dimensions: 5.98 x 9.01 x 0.50 inches

Eden 320W Tube Amplifier Head $999.99 Experience classic tube tone with this all-tube amplifier head that features a streamlined control set with an overdrive switch and a mid shift switch. The footswitch lets you change mute, overdrive and impedance settings while you play.

Randall George Lynch Series 100W Tube Amplifier $1149.99 Rock out with this tube amplifier that features 100 watts of all-tube power and specialty modules to help you achieve a distinctive sound.

Sovtek EL84EH Power Amplifier Vacuum Tube $11.95 An excellent way to recharge the tube amplifier in your guitar rig. Sovtek/Electro Harmonix EL84 (EL84EH) offers newfound power to give new life to your amplifier. A perfect replacement tube for VOX AC30, Peavey Classic 30 and Fender Blues Junior guitar amplifiers, the EL84EH power pentode from Sovtek/Electro Harmonix is a trusted tube that will keep your tube amplifier fired up from performance to performance.

Kustom Defender V5 Tube Guitar Combo Amplifier $169.95 The Defender V5 is a 5 watt tube combo amplifier with an 8 inch Kustom designed speaker! This Kustom guitar amplifier features great tones, and a simple linear tube circuit. The Defender V5 provides three speaker jacks for 4, 8 or 16 ohm operation. This Kustom also offers a 1/4 inch pigtail to the internal speaker if the use of an external speaker is desired. Get your hands on this great Kustom Defender V5 tube guitar combo amplifier! Kustom Defender V5 Tube Guitar Combo Amplifier Features: Powered by EL84 tubes 5 Watt Combo Amplifier 8 inch Kustom speaker Linear tube circuit Volume and Tone controls Three speaker jacks for 4, 8 or 16 lhm operation 1/4 inch pigtail to internal speaker Great for recording, rehearsal or plugging in your favorite stomp boxes

Jet City JCA22H Tube Guitar Amplifier Head $399.99 The Jet City JCA22H Tube Guitar Amplifier Head – You asked for it, and here it is! We took the legendary JCA20H and added a second, footswitch-accessible overdrive channel, along with a tube-buffered, serial effects loop. The JCA22H amplifier head is poised to change the amp world . . . again! It is elegance in design both inside and out, delivering unflinching reliability and everything you’d want in an amp. The JCA22H guitar amplifier head has full EQ control and separate preamp gain and master volumes, it can deliver spanky cleans all the way through saturated, Soldano high-gain overdrive. The Jet City JCA22H Tube Guitar Amplifier Head Features Two footswitchable overdrive channels Tube buffered, serial effects loop Full EQ control Separate preamp gain and master volumes Soldano high-gain overdrive

Hartke 1100W Bass Guitar Tube Amplifier Head $599.99 Tonal warmth and sonic power come together in this 1100W bass guitar tube amplifier head that features bass and treble shelving, mid-peak EQ controls and bright and limiter switches for easily customizing your sound.

Kustom Defender V15 Tube Guitar Combo Amplifier $269.95 The DefenderV15 is an 15-watt tube combo amplifier with a 10 inch Celestion speaker, great tones and professional features! In addition to the Volume and Tone controls, it provides a four position EQ low-frequency response control for American to British low end response and blended settings in-between. When you need a great quality amplifier, rely on Kustom Defender V15 tube guitar combo amplifier to pack the serious sound you desire! Kustom Defender V15 Tube Guitar Combo Amplifier Features: Powered by 2 EL84 tubes 15 Watts 10 inch Celestion speaker Four position EQ Low-frequency response control 1/4 Power switch Multiple impedance operation Transformer – tapped speaker-emulated XLR balanced line out with ground lift