Polymerase Chain Reaction (PCR) – Principle, Components, Steps, Types and Applications

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In molecular biology laboratories, only a small amount of deoxyribonucleic acid (DNA) may be obtained from a biological sample. For genetic analysis, it is often necessary to examine a particular region of the DNA. Polymerase Chain Reaction (PCR) is commonly used for this laboratory work. It was developed by Kary Mullis in 1983.

The DNA sample is mixed with the required PCR reagents in small reaction tubes. These tubes are then placed in a PCR machine, also called a thermal cycler (thermocycler). The machine controls the heating and cooling of the reaction tubes according to the programmed temperature conditions.

What is Polymerase Chain Reaction (PCR)?

Polymerase Chain Reaction (PCR) is an in vitro molecular biology technique used to amplify a specific sequence of deoxyribonucleic acid (DNA). It is a temperature-dependent enzymatic process.

A pair of short DNA primers binds to complementary sequences on the opposite strands of the template DNA. These primers define the region that will be amplified. DNA polymerase enzyme synthesizes new DNA strands. The heating and cooling are repeated in cycles, during which the DNA molecules produced in one cycle can again act as templates for the following cycle.

Conventional PCR amplifies DNA only. In the case of ribonucleic acid (RNA), the RNA is first converted into complementary DNA (cDNA) by the reverse transcriptase enzyme. The formed cDNA is then amplified by PCR. This is referred to as reverse transcription PCR (RT-PCR).

Purpose of PCR

The main purposes of PCR are as follows-

  • To amplify a specific segment of deoxyribonucleic acid (DNA) from a very small amount of starting sample. Millions of copies of the target DNA can be produced.
  • To detect the presence of a particular DNA sequence, even when it is present in a very minute amount in the sample.
  • To obtain sufficient copies of the selected DNA fragment for further molecular studies, such as DNA sequencing and gene cloning.
  • To identify genetic mutations and variations in the DNA sequence, including differences between alleles.

Principle of PCR

The principle of polymerase chain reaction (PCR) is based on the separation of deoxyribonucleic acid (DNA) strands, nucleic acid hybridization and the synthesis of new DNA strands by the process of DNA replication.

When the double-stranded DNA is heated, the hydrogen bonds between the complementary bases are broken, forming two single-stranded DNA templates. On lowering the temperature, the specific primers anneal to their complementary sites on the opposite DNA strands. The thermostable DNA polymerase enzyme then extends these primers by adding nucleotides at their 3′ ends, synthesizing new complementary DNA strands in the 5′→3′ direction. This enzyme can withstand repeated heating.

The newly synthesized DNA strands are again used as templates in the following cycle. During PCR, these reactions are repeated in a cyclic manner, increasing the number of target DNA molecules exponentially.

Theoretically, each cycle produces twice the number of DNA copies present at the beginning of that cycle.

The amplification of DNA can be calculated by the following equation-

Nₙ = N₀ × 2ⁿ

Here, N₀ is the initial number of target DNA copies, n represents the number of amplification cycles and Nₙ is the number of target DNA copies after n cycles. This calculation is based on 100% amplification efficiency.

In the actual PCR reaction, the DNA molecules are not always doubled in every cycle. As the reaction continues, the amount of available primers and nucleotides becomes limited. The DNA products also accumulate, and the activity of DNA polymerase may change. Amplification becomes slower during the later cycles.

The reaction eventually enters a plateau phase. At this stage, very little additional DNA is formed.

Components of PCR

Polymerase chain reaction (PCR) requires five main biochemical components for the amplification of deoxyribonucleic acid (DNA). These include template DNA, two oligonucleotide primers, thermostable DNA polymerase, deoxynucleotide triphosphates (dNTPs), and a suitable reaction buffer containing magnesium ions (Mg²⁺).

The following are the components required for conventional PCR.

1. Template DNA

  • Template DNA is the sample DNA containing the specific nucleic acid sequence that needs to be amplified during the PCR process.
  • It acts as the starting material for the formation of new complementary DNA strands. Different types of DNA can be used as templates, such as genomic DNA (gDNA), plasmid DNA, and complementary DNA (cDNA). The selected target sequence must be present in the template DNA.
  • The DNA sample should have suitable concentration and quality. Impurities and other substances that inhibit the DNA polymerase enzyme may interfere with the amplification reaction.

2. DNA Primers

  • Primers are artificially synthesized short single-stranded oligonucleotides, generally made up of about 18–30 nucleotides. They act as the starting point for the synthesis of new DNA strands by the DNA polymerase enzyme.
  • In conventional PCR, two primers are used, one forward primer and one reverse primer. The forward and reverse primers bind to their respective complementary sequences present on the opposite strands of the template DNA.
  • The primers are designed to bind at the two ends of the selected DNA region. The specific sequences and binding positions of these primers determine which segment of DNA will be amplified.
  • Each primer provides a free 3′ hydroxyl (3′-OH) end from which DNA polymerase begins adding nucleotides. The two primers should have compatible melting temperatures (Tₘ). Strong secondary structures and primer-dimer formation should be avoided during primer designing.

3. DNA Polymerase

  • DNA polymerase is an enzyme that catalyzes the synthesis of new complementary DNA strands by adding nucleotides sequentially to the 3′ end of an annealed primer. The sequence of nucleotides to be added is determined by the complementary bases present on the template DNA.
  • The enzyme used in PCR must be thermostable as the reaction involves repeated heating and cooling cycles. Taq DNA polymerase, obtained from the bacterium Thermus aquaticus, is one of the most commonly used DNA polymerase enzymes. It can withstand the high temperature of DNA denaturation and synthesizes new DNA strands efficiently at about 72°C.
  • The Taq DNA polymerase lacks 3′ to 5′ proofreading activity, due to which nucleotide incorporation errors are comparatively more frequent. Other thermostable enzymes, such as Pfu DNA polymerase, possess proofreading activity and have a lower error rate. The accuracy of DNA synthesis is referred to as fidelity. High-fidelity DNA polymerases are generally preferred when the DNA sequence needs to be amplified with fewer errors.

4. Deoxynucleotide Triphosphates (dNTPs)

  • Deoxynucleotide triphosphates are the nucleotide substrates used as building blocks for the synthesis of new DNA strands. There are four different types of dNTPs used in the PCR reaction, which are as follows-
    • Deoxyadenosine triphosphate (dATP)
    • Deoxythymidine triphosphate (dTTP)
    • Deoxyguanosine triphosphate (dGTP)
    • Deoxycytidine triphosphate (dCTP)
  • During the extension step, these nucleotides are sequentially added to the growing DNA strand by the DNA polymerase enzyme according to the complementary bases present in the template DNA.
  • All four dNTPs are required in a suitable and balanced concentration. Excessive concentration of dNTPs may inhibit the PCR reaction. On the other hand, insufficient nucleotides can limit the formation of new DNA strands.

5. PCR Buffer and Magnesium Ions

  • The PCR reaction is carried out in a suitable buffer system that maintains the required pH and ionic conditions for DNA polymerase activity. It is generally made up of buffering compounds, salts, and magnesium ions (Mg²⁺).
  • Magnesium ions act as an essential cofactor for the DNA polymerase enzyme. They are commonly supplied in the form of magnesium chloride (MgCl₂). The Mg²⁺ ions are required for the incorporation of dNTPs into the newly synthesized DNA strands.
  • The concentration of Mg²⁺ affects both the DNA polymerase activity and the binding of primers to the template DNA. At lower concentrations, DNA amplification may be reduced or completely inhibited. Excess Mg²⁺ can promote non-specific primer binding and decrease the fidelity of DNA synthesis.
  • The optimum buffer composition and Mg²⁺ concentration differ depending on the DNA polymerase and other components of the reaction mixture.

6. PCR Machine (Thermal Cycler)

  • The PCR machine, also known as a thermal cycler or thermocycler, is an electrical laboratory instrument used to regulate the temperature of the PCR reaction mixture. It provides the required heating and cooling conditions during the amplification process.
  • The machine is programmed to maintain different temperatures for a specified period. During denaturation, the temperature is increased to separate the double-stranded DNA into single strands. The temperature is then lowered for primer annealing and again adjusted for the extension of new DNA strands.
  • These temperature changes take place repeatedly for a certain number of cycles according to the pre-programmed conditions.
  • The thermocycler is a laboratory equipment used to carry out conventional PCR. It is not included among the five biochemical components added into the PCR reaction mixture.

Steps of PCR

Polymerase chain reaction (PCR) is a cyclic process in which a specific segment of deoxyribonucleic acid (DNA) is amplified by three main steps, denaturation, annealing, and extension. These three steps occur one after another in a single PCR cycle. The cycle is generally repeated about 25–35 times in conventional PCR.

Apart from these three steps, the reaction mixture is prepared first, followed by an initial denaturation before the amplification cycles begin. An optional final extension may also take place after the last cycle. The entire PCR procedure does not have a fixed “four-stage” classification.

The following are the steps involved in conventional PCR.

1. Preparation of PCR Reaction Mixture

  • It is the initial step before the actual PCR reaction takes place inside the thermocycler. The sample DNA is first extracted from the specimen or a previously prepared DNA sample can be used.
  • The PCR reaction mixture is made up of template DNA, forward and reverse primers, deoxynucleoside triphosphates (dNTPs), thermostable DNA polymerase, reaction buffer containing magnesium ions (Mg²⁺), and nuclease-free water. These components are added into a PCR tube.
  • The contents are mixed gently. The PCR tube is then closed and loaded into a pre-programmed thermocycler for the amplification process.

2. Initial Denaturation

  • Before starting the amplification cycles, the reaction mixture is first subjected to a high temperature of about 94–98°C for 1–5 minutes. At this temperature, the hydrogen bonds between the complementary bases of double-stranded DNA are disrupted. The two DNA strands separate from each other, forming two single-stranded DNA templates.
  • The initial denaturation time varies depending on the DNA polymerase used. In the case of some hot-start DNA polymerases, the initial heating also activates the enzyme.

3. Denaturation

  • It is the first step of each PCR cycle. The reaction mixture is again heated to about 94–98°C for 10–30 seconds, during which the double-stranded DNA molecules are separated into single strands by breaking the hydrogen bonds holding the complementary bases together.
  • The DNA molecules produced during the previous amplification cycles also get denatured. These single strands are used as templates for further DNA amplification.

4. Annealing

  • After denaturation, the temperature of the reaction mixture is reduced to about 50–65°C for 15–60 seconds. The forward and reverse primers now bind to their respective complementary sequences present on the two single-stranded DNA templates.
  • The temperature required for annealing is based on the melting temperature (Tₘ) of the primers. It is generally maintained a few degrees below the lower primer Tₘ.
  • Each annealed primer provides a free 3′ end from which the DNA polymerase enzyme can begin synthesizing a new DNA strand.

5. Extension (Elongation)

  • In this step, the temperature is again increased to about 68–72°C. When Taq DNA polymerase is used, the temperature is commonly maintained at 72°C.
  • The DNA polymerase enzyme adds dNTPs sequentially to the 3′ end of the annealed primer. A new complementary DNA strand is formed in the 5′ to 3′ direction.
  • The time required for elongation depends on the length of the target DNA and the activity of DNA polymerase. Generally, about 1 minute per kilobase of target DNA is used with conventional Taq polymerase, whereas other polymerases may require shorter extension periods.
  • Extension is the third and last step within a PCR cycle. The newly formed DNA molecules can then act as templates in the subsequent cycle, allowing further amplification of the selected DNA segment.

6. Final Extension

  • After the completion of the required amplification cycles, the reaction mixture may be subjected to an additional extension period at about 72°C for 5–10 minutes. During this process, DNA polymerase completes the synthesis of DNA strands that remained incompletely extended during the previous cycles.
  • It is an optional step. When included, the final extension is the last DNA synthesis stage of the PCR process.
  • In PCR reactions where final extension is not included, the extension step of the last amplification cycle becomes the final stage of DNA synthesis.
  • Some thermocycler programs also include a final holding temperature of about 4°C until the PCR tubes are removed from the machine. The exact temperature, duration, and number of cycles differ depending on the DNA polymerase, primer Tₘ, and length of the target DNA.

Analysis of PCR Products

After completion of conventional polymerase chain reaction (PCR), the amplified product is examined to check whether the expected deoxyribonucleic acid (DNA) fragment has been formed. This is generally done by agarose gel electrophoresis. The size and number of DNA bands are observed along with the control reactions.

Agarose Gel Electrophoresis of PCR Products

Agarose gel electrophoresis is commonly used for the analysis of PCR products. In this method, a small portion of the PCR product is mixed with loading dye and loaded into the wells of an agarose gel. A DNA ladder containing fragments of known sizes is also loaded in a separate well.

When an electric current is applied, the negatively charged DNA fragments move towards the positive electrode. Smaller DNA fragments migrate faster and travel a greater distance through the gel compared to larger fragments. The separated DNA bands are then visualized using a suitable nucleic acid stain under ultraviolet (UV) or blue light, depending on the stain used.

The position of each DNA band is compared with the DNA ladder to determine its approximate size in base pairs (bp). The expected size of the PCR product is known from the positions of the forward and reverse primers on the target DNA.

Interpretation of PCR Bands and Controls

The PCR results are interpreted by observing the presence, absence, size and number of DNA bands on the agarose gel. The following are the common observations during PCR product analysis.

  1. Expected DNA Band- The presence of a distinct DNA band at the expected size indicates that amplification has produced a fragment of the required approximate length. Usually, a single clear band is expected when PCR is designed to amplify one specific DNA fragment. However, the band size alone cannot confirm the exact nucleotide sequence. DNA sequencing may be required for confirmation.
  2. Absence of DNA Band- If no band is observed at the expected position, the required PCR product was not detected. It may occur due to insufficient template DNA, poor DNA quality, PCR inhibitors or unsuitable reaction conditions. Very small amounts of amplified DNA may also remain below the detection limit of the gel.
  3. Nonspecific Amplification Bands- Sometimes two or more bands of different sizes appear on the gel. These additional bands are generally produced due to nonspecific binding of primers to other regions of DNA. In some cases, the amplified DNA may appear as a smear rather than distinct bands.
  4. Primer-Dimers- Small DNA bands near the bottom of the gel may indicate the formation of primer-dimers. These are produced when primers bind to each other and are extended by DNA polymerase. Primer-dimers are generally much smaller than the expected PCR product.
  5. Positive Control- A positive control contains a known DNA template that can be amplified by the PCR reaction. It should produce a band of the expected size. If no band appears in the positive control, there may be some problem with the PCR reagents, reaction setup or thermal cycling conditions. In this case, the absence of a band in the test sample cannot be reliably interpreted.
  6. No-Template Negative Control- The no-template control (NTC) contains all the PCR reagents except the template DNA, which is replaced with nuclease-free water. Normally, no target-sized DNA band should be present in this lane. A band at the expected product size may indicate contamination of the reaction mixture with DNA. A very small band may also appear due to primer-dimer formation.

Types of PCR

Polymerase chain reaction (PCR) has several modified forms based on the method of amplification, type of nucleic acid sample and purpose of the reaction. Some of the important types of PCR are as follows-

  1. Conventional PCR (End-point PCR)- It is the basic type of PCR used to amplify a specific segment of deoxyribonucleic acid (DNA). The amplified products are generally analyzed by agarose gel electrophoresis after completion of the reaction.
  2. Real-Time PCR (qPCR)- Real-time or quantitative PCR is a technique in which the amplification of DNA is monitored during each cycle using fluorescent dyes or probes. It is used for the quantification of target DNA.
  3. Reverse Transcription PCR (RT-PCR)- It is used for the amplification of ribonucleic acid (RNA) targets. First, the RNA is converted into complementary DNA (cDNA) by reverse transcriptase enzyme. The cDNA is then amplified by PCR. This reaction can be performed by one-step or two-step methods.
  4. Reverse Transcription Quantitative PCR (RT-qPCR)- It combines reverse transcription with real-time PCR. The RNA is first converted into cDNA, which is then amplified and measured using fluorescence. It is commonly used for gene expression analysis and quantification of RNA targets.
  5. Digital PCR (dPCR)- In this method, the PCR mixture is divided into a large number of small individual reactions. After amplification, the positive and negative reactions are counted to determine the number of target DNA copies. It allows absolute quantification without a standard curve. Droplet digital PCR (ddPCR) is one of its common forms, where the reaction mixture is divided into thousands of small droplets.
  6. Multiplex PCR- It is a type of PCR used to amplify two or more different DNA sequences in a single reaction mixture. Multiple pairs of primers are added to the same reaction tube.
  7. Nested PCR- Two successive amplification reactions are performed using two sets of primers. In the first reaction, a larger DNA fragment is amplified. The product is then used as a template for the second reaction with another pair of primers that bind within the first amplified region. It increases the specificity and sensitivity of PCR.
  8. Hot-Start PCR- In this method, DNA polymerase activity is blocked at lower temperatures and activated during the initial heating step. It reduces nonspecific amplification and primer-dimer formation during reaction preparation.
  9. Touchdown PCR- The annealing temperature is initially kept higher than the usual annealing temperature and gradually reduced during successive cycles. This method is used to improve the specificity of primer binding.
  10. High-Fidelity PCR- It uses DNA polymerases with high accuracy, commonly having proofreading activity, to reduce errors during DNA amplification. It is mainly used for DNA cloning, sequencing and other techniques where accurate DNA copies are required.
  11. Long-Range PCR- This technique is used for the amplification of comparatively long DNA fragments. Special DNA polymerases or enzyme mixtures and optimized reaction conditions are used to amplify large DNA regions.
  12. Allele-Specific PCR- It uses primers designed to selectively amplify a particular allele of a gene. The amplification depends on the matching of the primer with the target sequence, especially near its 3′ end. It is used for detecting known mutations and single nucleotide polymorphisms (SNPs).
  13. Methylation-Specific PCR (MSP)- It is used to determine the methylation status of specific DNA regions. The DNA is first treated with bisulfite, which converts unmethylated cytosines into uracil while methylated cytosines are largely retained. Different primer sets are then used to amplify methylated and unmethylated sequences.
  14. Asymmetric PCR- In this type of PCR, unequal concentrations of forward and reverse primers are used. After the limiting primer is exhausted, one strand of DNA is produced in excess. It is used for the preparation of single-stranded DNA.
  15. Inverse PCR- It is used to amplify unknown DNA sequences located next to a known DNA region. The DNA is usually digested with restriction enzymes and circularized by ligation. Primers directed outward from the known sequence are then used for amplification.
  16. Colony PCR- Bacterial colonies are used directly as the source of template DNA without requiring a separate plasmid purification step. It is commonly used to screen bacterial colonies for the presence of a desired DNA insert.
  17. Overlap Extension PCR- This method is used to join two or more DNA fragments having overlapping sequences. The overlapping regions anneal with each other and are extended by DNA polymerase to form a longer DNA fragment. It is also used for introducing specific mutations into DNA sequences.
  18. Random Amplified Polymorphic DNA PCR (RAPD-PCR)- It uses short primers of arbitrary sequences to amplify different regions of genomic DNA. Prior knowledge of the target DNA sequence is not required. The amplified DNA fragments produce banding patterns which are used for genetic diversity analysis and DNA fingerprinting.
  19. In Situ PCR- The amplification of target nucleic acid sequences takes place within fixed cells or tissue sections. It is used to detect and localize specific DNA sequences while retaining their position in the cells or tissues.
  20. Gradient PCR- Different annealing temperatures are tested simultaneously using a gradient thermal cycler. It is mainly used to determine the suitable annealing temperature for a particular primer pair and optimize PCR conditions.
  21. High-Resolution Melting PCR (HRM-PCR)- It combines PCR amplification with high-resolution melting analysis of the amplified DNA. After amplification, the DNA is gradually heated and changes in fluorescence are measured to study its melting behavior. Differences in melting curves are used to identify sequence variations.
  22. Co-amplification at Lower Denaturation Temperature PCR (COLD-PCR)- It is a modified PCR technique used for the enrichment of low-abundance DNA mutations. A carefully selected critical denaturation temperature is used to preferentially amplify certain sequence variants from a mixture of normal and mutant DNA.

Applications of PCR

Polymerase chain reaction (PCR) is widely used in different fields of molecular biology, medicine, agriculture and biotechnology. Some of its important applications are as follows-

  • PCR is used for the detection and identification of disease-causing microorganisms, including bacteria, viruses and fungi, by amplifying their specific nucleic acid sequences.
  • It is used for the identification and classification of microorganisms based on their genetic characteristics. Different bacterial species and strains can also be distinguished using specific genetic markers.
  • Mutations associated with genetic disorders such as sickle cell anemia, thalassemia and cystic fibrosis can be detected using PCR-based methods. It is also used in prenatal genetic testing for selected inherited disorders.
  • In cancer research, PCR is used to detect specific gene mutations and genetic alterations associated with the development of cancer.
  • PCR is widely used in forensic science for DNA fingerprinting, identification of individuals, criminal investigations and paternity testing. For this, selected short tandem repeat (STR) regions of deoxyribonucleic acid (DNA) are amplified and compared.
  • Specific DNA fragments are amplified by PCR and used for gene cloning and genetic engineering. It is also used to check the presence of the desired gene in recombinant DNA molecules.
  • For DNA sequencing, PCR is used to produce multiple copies of a selected DNA fragment before determining its nucleotide sequence.
  • PCR is used for the study of gene expression by first converting messenger ribonucleic acid (mRNA) into complementary DNA (cDNA) through reverse transcription. The amount of gene expression can be measured using quantitative reverse transcription PCR.
  • The quantity of a specific DNA sequence can be measured by quantitative PCR (qPCR). It is used for determining gene copy numbers and measuring viral DNA loads.
  • In agriculture, PCR is used to identify bacterial, fungal and viral pathogens responsible for different plant diseases.
  • PCR-based genetic markers are used in plant and animal breeding for the identification and selection of individuals having desired genetic characteristics.
  • In food industries, PCR is used to detect foodborne pathogens and identify the animal or plant species present in food products. It is also used for detecting certain types of food adulteration.
  • PCR is employed in environmental microbiology to study microbial diversity in soil, water and other environmental samples. It allows the detection of microorganisms that are difficult to grow under laboratory conditions.
  • DNA obtained from fossils and ancient biological remains can be amplified using PCR for studying ancient organisms, genetic relationships and evolutionary history.

Advantages of PCR

  • Polymerase chain reaction (PCR) is a very rapid method for producing multiple copies of a specific deoxyribonucleic acid (DNA) segment. Millions of copies can be produced within a few hours.
  • It has high sensitivity and can amplify a very small amount of DNA, even when only a few copies of the target sequence are present in the sample.
  • It has high specificity as the primers are designed to bind to particular regions of the template DNA. The specificity depends on the primer sequences and reaction conditions.
  • PCR does not require the cultivation of microorganisms or cloning of DNA into living cells before amplification.
  • Its principle and procedure are comparatively simple. The heating and cooling cycles are automatically regulated by a thermal cycler. Multiple samples can also be processed in a single run.
  • DNA obtained from different biological materials such as blood, saliva, tissues and microbial samples can be used for amplification. In some cases, highly purified DNA is not required, provided the sample does not contain inhibitory substances.
  • It provides sufficient amplified DNA for various molecular techniques like gene cloning, DNA sequencing, mutation analysis and DNA fingerprinting.
  • Modified forms of PCR can also be used for the quantitative analysis of nucleic acids. In quantitative PCR (qPCR), the amplification is monitored during the reaction to estimate the initial amount of target DNA.

Limitations of PCR

  • Prior information regarding the target deoxyribonucleic acid (DNA) sequence is generally required for designing specific primers. Changes in the primer-binding regions may prevent the amplification of the target DNA.
  • Even a very small amount of contamination with foreign DNA or previously amplified PCR products can lead to unwanted amplification and false-positive results.
  • Several substances present in the sample, such as hemoglobin, humic acids and chemicals remaining after DNA extraction, can inhibit DNA polymerase activity. This may reduce amplification efficiency or produce false-negative results.
  • The quality and quantity of the template DNA can affect the PCR reaction. Degraded DNA or a very low amount of template may fail to produce the expected amplification product.
  • There is a chance of nonspecific amplification if the primers bind to other similar DNA sequences. Primer-dimer formation can also occur. These reactions may reduce the amount of the desired PCR product.
  • DNA polymerase may introduce errors during DNA synthesis. The error rate depends on the polymerase used, and some enzymes such as Taq DNA polymerase lack proofreading activity.
  • PCR is highly dependent on proper reaction conditions, including annealing temperature, magnesium ion concentration and primer concentration. Incorrect conditions may reduce the efficiency and specificity of amplification.
  • It is difficult to amplify very long DNA fragments or regions having high guanine-cytosine (GC) content using conventional PCR. Such DNA regions may require special polymerases and modified reaction conditions.
  • PCR cannot directly amplify ribonucleic acid (RNA). The RNA must first be converted into complementary DNA (cDNA) using reverse transcriptase before amplification.
  • Conventional endpoint PCR is mainly used for qualitative analysis. It cannot reliably determine the initial quantity of target DNA from the final PCR product alone.
  • The presence of a DNA band at the expected position does not always confirm the exact identity of the amplified sequence. Additional analysis, such as DNA sequencing, may be required.
  • PCR cannot distinguish between living and dead microorganisms when amplifiable DNA is present in both. DNA from dead microorganisms may also produce an amplification product.

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