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Cite this page: Shackelford R. PCR overview. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/MolecularPCRoverview.html. Accessed June 20th, 2021.
Basic mechanism and protocol

Components and reagents of a typical PCR
  • DNA template: most often fractionated whole genomic DNA which likely contains the sequence to be amplified; may be single or double stranded DNA (Wikipedia: DNA Replication [Accessed 4 June 2018])
  • 2 oligonucleotide primers: single stranded DNA, often 20 - 30 base pairs (bp) which are complementary to the 3' ends of the sense and antisense strands of the DNA sequence to be amplified
  • DNA polymerase: usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98 °C) and can function at a temperature optimum of about 70 °C (see also below)
  • Buffer solution and monovalent and divalent cations: includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity
  • Deoxynucleoside triphosphates: provides the energy for polymerization and the building blocks for DNA synthesis

Protocol
  • PCR has many variations but this protocol is typical
  • Most protocols involve 15 - 40 repeated thermocycles, with reaction volumes of 5 - 100 μL
  • Oligonucleotides, nucleoside triphosphates, DNA with the sequence to be amplified, buffer and a DNA polymerase are mixed together and subjected to different temperatures
  • Often a "water only" control, with all required PCR reagents but water instead of DNA to be amplified, is simultaneously run to look for DNA contamination; this control should show no amplification; if it shows amplification, it indicates that a contaminating DNA is present
  • Exact temperature and length of each PCR step in the thermocycling reaction varies according to the melting temperature of the oligonucleotides used and the length of the elongation step

  1. Initialization:
    • Reaction is heated to 94 - 96 °C for 30 seconds to several minutes to completely denature the nucleic acids, which lowers nonspecific nucleic acid interaction and inappropriate priming events
    • Additionally, some DNA polymerases require heat activation and require temperatures as high as 98 °C for several minutes to both denature the nucleic acids and activate the DNA polymerase
    • Typically, this step is done only once in a PCR reaction

  2. Denaturation:
    • This is the first step in the thermocycling reactions that will be repeated
    • It causes nucleic acid denaturation or separation of the oligonucleotide primers from the template DNA to be amplified, resulting in all the DNA in the reaction to be single stranded
    • Usually this step involves heating the reaction to 94 - 98 °C for 15 - 30 seconds

  3. Annealing:
    • Reaction temperature is rapidly lowered to 50 - 64 °C for 20 - 40 seconds
    • Since the template DNA and oligonucleotides are moving randomly in the reaction mix, the lower temperature will allow the template DNA and oligonucleotides to form Watson-Crick base pairs, resulting in double stranded DNA
    • Annealing temperature is relatively high, allowing only the most stable and therefore, specific double stranded DNA structures to form
    • Double stranded DNA structures which have lower stability due to a few or many non-Watson-Crick base pairings are unlikely to form or initiate a nonspecific DNA synthesis
    • Additionally, the DNA polymerase binds the double stranded DNA at this step and initiates DNA synthesis

  4. Elongation:
    • Also known at extension, this step usually occurs at 72 - 80 °C (most commonly 72 °C)
    • Length of the step depends on the length of the DNA being synthesized
    • Under optimal conditions, DNA polymerase will add about 1,000 bp/minute
    • Higher temperature used in elongation compared to annealing again lowers nonspecific oligonucleotide template interactions, increasing the specificity of the reaction
    • In this step, the DNA polymerase uses the nucleoside triphosphates to synthesize DNA in the 5' to 3' direction
    • After the first few rounds of synthesis, the length of the amplified DNA is limited by where each oligonucleotide binds, thus nearly all amplified DNA will be as long as the distance between two oligonucleotides on the template DNA
    • With each of the above temperature cycles (steps 2 - 4 above), the amount of DNA synthesized is doubled, resulting in exponential DNA amplification
    • Most PCR reactions do 15 - 40 cycles of steps 2 - 4

  5. Final elongation:
    • This step in not always performed
    • Reaction is held at 70 - 74 °C for several minutes after the last PCR to allow any remaining single stranded DNA to be fully extended

  6. Final hold:
    • Reaction is complete and the resulting amplified nucleic acids are held at a low temperature (~4 °C) until analysis

  7. Analysis:
    • This step can involve many different methods
    • Often completed PCRs are analyzed by gel electrophoresis, where the amplified DNA is run into a gel and visualized via ethidium bromide staining seen under UV light
    • Usually a "water blank" reaction and a DNA ladder are also run into the gel as controls for possible DNA contamination and for molecular weight identification
Taq polymerase
  • Initially, the Klenow fragment from the E. coli DNA polymerase I was used in PCR reactions
  • While the Klenow fragment worked in PCR, it rapidly denatured at the 90 °C or higher temperatures required for denaturation, requiring the addition of new Klenow polymerase after every denaturation step
  • The need to open the reaction chamber with every PCR thermocycle made PCR cumbersome and greatly increased the chances for contamination with unwanted DNA
  • The solution to this problem came from the discovery and cloning of a thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus
  • The polymerase, known as the Taq polymerase, has many attributes that are well suited for PCR:
    1. Enzyme works best at 75 - 80 °C, allowing the elongation step to occur at temperatures which make non-Watson-Crick base paring a rare event
    2. Its half life at 95 °C is over 40 minutes
    3. At 72 °C it can add 1,000 nucleoside triphosphates to a growing DNA strand
  • Most PCR reactions done with Taq can be completely closed through all the thermocycles
  • Taq lacks 3' to 5' exonuclease proofreading activity, allowing a roughly 1 per 9,000 error rate in nucleoside triphosphate incorporation
  • Additionally, Taq often adds an adenine to the end of its polymerase reaction (sometimes used to facilitate TA cloning)
  • Taq was named "The Molecule of the Year" by Science in 1989
Reaction stages
  • Thermocycles in PCR can be divided into three stages
  • Understanding where each of these stages occurs in each PCR reaction is important, especially for the analysis of end point PCR reactions

Exponential amplification:
  • Initially, all the reagents employed (primers and nucleoside triphophates) in PCR are abundant and the Taq polymerase is fully functional
  • Therefore, the initial phases of PCR are characterized by near 100% efficient DNA amplification, with the amount of replicated DNA doubled per thermocycle

Leveling off stage:
  • In later thermocycles, the Taq polymerase begins to lose its activity and the concentration of primers and nucleoside triphophates become rate limiting, hence the reaction rate significantly slows

Plateau:
  • Eventually, no new DNA amplification occurs due to exhaustion of the reagents and loss of enzyme activity
Thermocycling machines
  • Early PCR protocols required that each reaction be manually transferred to water baths of different temperatures at each temperature change
  • This problem was eliminated with the development of PCR thermocyclers, which rapidly change reaction temperatures without the need for manual transfers
  • The first machine for this purpose, "Mr. Cycle," was developed by Cetus Corporation
  • Since then, many different thermocyclers have been developed; most can be programmed to allow any desired temperature and time for each PCR cycle
  • More recent PCR machines can analyze multiple parallel real time PCR reactions and analyze the data via various computer algorithms
Methylation specific
  • In mammalian cells, genes are often regulated by the addition of a -CH3 group (methylation) to specific promoter cytosine moieties that are followed by a guanosine
  • The methylation of this dinucleotide, called a "CpG island," results in inhibition of gene transcription and hence repression of gene expression
  • Interestingly, cytosine methylation is very stable and heritable and is passed through cell division, making cytosine methylation a major mechanism for gene imprinting
  • Additionally, aberrant cytosine methylation is commonly seen in malignancies, where the expression of tumor suppressor genes is lowered or completely blocked by this mechanism

Detection:
  • Cytosine methylated DNA can be distinguished from cytosine unmethylated DNA by in vitro sodium bisulfite treatment, which converts unmethylated cytosine into uracil but does not change methylated cytosine
  • Using PCR with primers that bind either the modified or unmodified CpG containing sequences can be used to look for the cytosine to uracil modification: in an unmodified promoter sequence, the cytosines would bind a primer with guanosines but with bisulfite modification, the promoter uracils would bind a primer with thymines
  • Running separate PCR reactions with each set of primers (one for methylated DNA and one for nonmethyated DNA), would easily reveal the presence or absence of cytosine methylation
  • When analyzed by gel electrophoresis, followed by ethidium bromide staining and UV visualization, unmethylated DNA is detected when cytosine binds to a primer with guanosine primer and methylated DNA is detected when cytosine binds to a primer with thymine
  • Although this technique is commonly used, it has disadvantages of:
    1. Bisulfite treatment destroys about 90% of the DNA
    2. Some of the reagents are toxic
Multiplex
  • In multiplex PCR, several discrete sequences are amplified simultaneously in the same reaction using multiple primer pairs
  • The technique is used to verify that an amplifiable sequence is present in a specific sample or to amplify multiple sequences within one reaction
  • Usually the sequence being searched for is coamplified with a known housekeeping gene, which is always present and easily amplified
  • If only the housekeeping gene is amplified, then the other sequence is absent from the original sample
  • If neither sequence is amplified, then a PCR inhibitor is likely present
  • Up to 20 different reactions can be run simultaneously, thus lowering the amount of sample used, reducing the reagents consumed and collecting far more information per reaction, while simplifying data analyses
  • This technique is widely used in forensics, tissue identification, detection of different species of viral nucleic acids within cerebrospinal fluid and transplantation engraftment studies, where it can use single nucleotide polymorphisms and other variation in DNA sequences to identify tissue sample origins
  • The main disadvantages are the difficulty, sometimes extreme, in optimizing the PCR reaction so that each sequence amplification is roughly as efficient as the other reaction within the same reaction chamber and designing primers that will not interact with each other (i.e. primer dimmers), resulting in nonspecific amplifications
Nested
  • Nested PCR uses two sets of primers, one set is internal to or "nested within" with other set
  • The first or "outer" pair of primers amplifies the target sequence; then a dilution is made of the reaction and the internal or nested primers are used to continue the amplification
  • Alternatively, the technique can be "seminested" where one original PCR primer and one internal primer are used after the first amplification
  • Both techniques will generate an amplified sequence shorter than the first amplified sequence
  • If the first reaction is amplified nonspecifically, then it is unlikely that the internal amplification would also proceed
  • Thus, this technique increases the specificity and sensitivity of the PCR reaction
  • A drawback of this technique is that the addition of new primers after the first amplification round increases the chances of nonspecific contamination; many clinical labs avoid this technique for this reason
Reverse transcriptase
  • In 1977, RNA levels were first routinely quantified via northern blotting
  • Northern blotting uses denaturing gel electrophoresis blotting with labeled DNA probes, extensive washing steps, followed by multiple film exposures to insure that an exposure within the linear range of the film is achieved
  • Northern blotting is relatively complex and time consuming and requires a large amount of RNA, making the examination of many different transcripts difficult
  • Additionally, northern blotting is poor at detecting low abundance RNA species
  • With the discovery of reverse transcriptase, which converts RNA to DNA, PCR could be used to amplify very low levels of RNAs (reverse transcriptase PCR or RT-PCR)
  • RNA could be converted by reverse transcriptase into a cDNA in one step and then PCR could amplify the cDNA
  • With minor modifications, this technique can be made real time, allowing the comparison of the relative abundance of different RNA species
  • Compared to northern blotting, RT-PCR has several advantages:
    1. It requires little post-PCR processing, unlike the cumbersome multisteps of northern blotting
    2. It can analyze a wide range (> 107 fold) of difference in RNA quantities, unlike northern blotting
    3. The assay is far more quantitative than northern blotting, allowing more accurate measurements of RNA species amounts

Other advantages of RT-PCR:
  • There are many diseases in which RNA analysis is preferable to DNA analysis
  • In chronic myelogenous leukemia (CML), analysis of the BCR-ABL fusion gene transcript is fairly straight forward with RT-PCR, since designing oligonucleotide primers which can span the region where the two genes are fused in not difficult
  • However, analysis of the same fusion region in DNA would require often impossibly long PCR reactions, as the breakpoint regions within the DNA sequence can be great distances from each other and also occur at different point within the BCR gene
  • Thus, DNA analysis by PCR would require the design of many different primers and probes to cover the different breakpoints in the chronic myelogenous leukemia the BCR-ABL gene fusion gene
  • RT-PCR requires far fewer, making the analysis far simpler
  • Often the meaning of the abbreviation "RT-PCR" is ambiguous and can mean either reverse transcriptase PCR or real time PCR; when writing about these techniques, you should clearly define exactly what technique is being discussed
  • An essential step in RT-PCR is the priming of the reverse transcriptase reaction; this is usually done by one of three methods below

Random primers:
  • Usually this method uses random heximer primers
  • All or most of the RNA templates within a sample are primed and amplified at multiple origins
  • Advantages:
    • Random primer mix can prime any RNA mix
    • There is no need to design specific probes
    • Works well for applications not requiring exact calculations of the abundance of a specific RNA
  • Disadvantages:
    • It preferentially amplifies ribosomal RNA, distorting the actual abundance of less common RNAs in the subsequent PCR reactions (the distortion of actual RNA abundance can be as high as 19 fold)
    • Random primers give a narrower linear range in PCR amplification curves than do reactions primed by sequence specific primers

Oligo-dT priming:
  • This priming method is more specific than the random method and is best used when one wants obtain a faithful representation of the mRNA pool and when one has a limited amount of mRNA to be amplified
  • Disadvantages include that it does not work if:
    • mRNA to be amplified is fragmented
    • mRNA has extensive secondary structure that may inhibit PCR
    • Later DNA amplification 5' priming site is very far away from the end of the oligo-dT priming site, such as in mRNAs with very long untranslated 3' sequences
    • mRNA has multiple splice variants, which differ at the 5' mRNA end and may not bind a specific 5' primer

Target specific primers:
  • Target specific primers give the most specific cDNA, providing the greatest sensitivity for quantitative assays
  • Main problem with this method is that it requires specific RNA amplifying primers and separate reactions for each target and therefore cannot be used on the same RNA sample; thus, it is a poor way to make cDNA when one has limited RNA
  • Multiplex priming is possible but requires very careful primer design
Real time
  • Also called quantitative real time PCR, this technique simultaneously amplifies and quantifies the amount of a specific DNA sequence during amplification by comparing the sample being amplified to standardized controls, hence the term "real time"
  • The quantification may be the relative amount of DNA compared to a known standard or amplification copy number
  • Initially, agents such as ethidium bromide were added to PCR reactions to quantify DNA during amplification
  • Ethidium bromide can detect increasing DNA concentrations in closed tube PCR reactions following excitation at 254 nm UV light
  • However, ethidium bromide has several drawbacks, including:
    1. It is a powerful mutagen
    2. Its fluorescence is not specific for double stranded DNA
    3. It can inhibit DNA polymerase activity

SYBR green:
  • In contrast to ethidium bromide, is specific for double stranded DNA or for the amplified DNA sequence
  • A cyanine dye which specifically binds double stranded DNA, with a double stranded DNA bound fluorescence quantum yield over 5x greater than the DNA / ethidium bromide complex; absorbs at 488 nm (blue) and strongly emits at 522 nm (green)
  • To quantify DNA amplification with SYBR green, the dye is added to the PCR reaction and the relative sample fluorescence is measured and compared to standard controls upon completion of each thermocycle (i.e. when double stranded DNA has formed with the reaction tube)
  • Since the concentration of double stranded DNA is measured, only the relative amount of DNA can be determined, not the exact amplification number
  • While this technique works well, it has the drawback that SYBR green binds to all double stranded DNA equally; thus any nonspecific double stranded DNA in the reaction (i.e. primer dimers) is also measured
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