Polymerase chain reaction & types.pdf

Module 2- Laboratory Methodologies Polymerase Chain Reaction Devised by KARY MULLIS 1988 Kary received a Nobel Prize in chemistry in 1993, for his invention of the Polymerase Chain Reaction. Polymerase chain reaction results in the selective amplification of a chosen region of a DNA molecule. E...

Module 2- Laboratory Methodologies Polymerase Chain Reaction Devised by KARY MULLIS 1988 Kary received a Nobel Prize in chemistry in 1993, for his invention of the Polymerase Chain Reaction. Polymerase chain reaction results in the selective amplification of a chosen region of a DNA molecule. Exploits the features of DNA replication. Taq polymerase uses a Single Strand template to synthesize a complementary strand. DNA Replication is Complementary Semiconservative Synthesis in 5’ to 3’ direction Can be duplicated in vitro with template+ DNA polymerase+ 4 nucleotides+ primer+ buffer with repeated denaturing and annealing In 1960s Biologist Thomas Brock discovered thin pink threads of bacteria thriving in one of the Yellowstone National Park’s hot springs HISTORY OF PCR at temperatures above 80 degrees able to withstand such extreme conditions. He named it Thermus aquaticus – Taq for short Kary B. Mullis Kary B Mullis Revolutionized Molecular Biology by devising the brilliant PCR technology in 1988 and won the Nobel prize in 1993. Science journal named Taq polymerase its first ‘Molecule of the Year’ in 1989 Mullis’ original technique used DNA polymerase from E. coli which was heat sensitive. More enzyme had to be added in between each heat cycle which was as wasteful and inconvenient. Then Mullis and his colleagues at the Cetus Corporation hit on the idea of using the DNA polymerase from Taq which was heat stable. By the early 90s, PCR was being used across virtually every area of molecular biology, and has grown into a multi-billion dollar industry. YELLOW STONE NATIONAL PARK Requirements of PCR DNA template containing sequence to be amplified. 2 oligonucleotide primers. Taq polymerase Mixture of 4 deoxynucleotide precursors. TAQ Polymerase: Originally E coli Polymerase was used in PCR. Temperature-resistant DNA polymerase from Thermus aquaticus Thomas Brock isolated T aquaticus. Chein et al isolated the polymerase in 1976. Allowed automation of PCR Thermal cyclers Fidelity of Taq pol. In vitro 2 in 104 wrong nucleotides are incorporated as proof reading is absent. PCR is a three-step process that is carried out in repeated cycles. The initial step is the denaturation, or separation, of the two strands of the DNA molecule. This is accomplished by heating the starting material to temperatures of about 95 °C (203 °F). Each strand is a template on which a new strand is built. In the second step the temperature is reduced to about 55 °C (131 °F) so that the primers can anneal to the template. In the third step the temperature is raised to about 72 °C (162 °F), and the DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about five minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA. The polymerase chain reaction amplifies DNA regions of known sequences. To amplify a specific region of DNA, an investigator will chemically synthesize two different oligonucleotide primers complementary to sequences flanking the region of interest. The complete reaction is composed of a complex mixture of double-stranded DNA (usually genomic DNA containing the target sequence of interest), a stoichiometric excess of both primers, the four deoxynucleoside triphosphates, and a heat-stable DNA polymerase known as Taq polymerase. During each PCR cycle, the reaction mixture is first heated to separate the strands and then cooled to allow the primers to bind to complementary sequences flanking the region to be amplified. Taq polymerase then extends each primer from its 3’end, generating newly synthesized strands that extend in the 3’direction to the 5’ end of the template strand. During the third cycle, two double-stranded DNA molecules are generated equal in length to the sequence of the region to be amplified. In each successive cycle the target segment, which will anneal to the primers, is duplicated, and will eventually vastly outnumber all other DNA segments in the reaction mixture. Successive PCR cycles can be automated by cycling the reaction for timed intervals at high temperature for DNA melting and at a defined lower temperature for the annealing and elongation portions of the cycle. A reaction that cycles 20 times will amplify the specific target sequence 1-million-fold. Essential components of PCR Thermostable polymerase to catalyze template-dependent synthesis of DNA. A wide choice of enzymes is now available that vary in their fidelity, efficiency, and ability to synthesize large DNA products For routine PCRs, Taq polymerase remains the enzyme of choice. Thermostable DNA polymerases are isolated from two classes of organisms: the thermophilic and hyperthermophilic eubacteria Archaebacteria, whose most abundant DNA polymerases are similar to DNA polymerase I of mesophilic bacteria Taq (T. aquaticus) DNA polymerase, the first isolated and best understood of the thermostable DNA polymerases, remains the workhorse of PCR in most laboratories. However, when greater fidelity is required, when the length of the target amplicon exceeds a few thousand bases, or when cloning mRNA by reverse transcriptase-PCR (RT-PCR), other thermostable enzymes may have significant advantages. The choice among enzymes should be determined by the purpose of the experiment. For example, if the goal is to make faithful copies of a gene, an enzyme with proofreading function is required, whereas if the goal is to clone an amplified product, an enzyme that generates blunt ends may be of advantage. Essential components of PCR A pair of synthetic oligonucleotides to prime DNA synthesis. Design of oligonucleotide primers affects the efficiency and specificity of the amplification reaction. Oligonucleotide primers synthesized on an automated DNA synthesizer can generally be used in standard PCRs without further purification. However, amplification of single-copy sequences from mammalian genomic templates is often more efficient if the oligonucleotide primers are purified by chromatography on commercially available resins or by denaturing polyacrylamide gel electrophoresis. TYPES OF PRIMES USED IN PCRs The common Tm formulas for calculating the theoretical Tm of an oligo-The simplest formula: Tm = 4 x (number of G’s and C’s in the primer) + 2 x (number of A’s and T’s in the primer) °C Essential components of PCR Deoxynucleoside triphosphates (dNTPs). Standard PCRs contain equimolar amounts of dATP, dTTP, dCTP, and dGTP Divalent cations. All thermostable DNA polymerases require free divalent cations — usually Mg2+ — for activity. Some polymerases will also work, albeit less efficiently with buffers containing Mn2+. Buffer to maintain pH. Tris-Cl, adjusted to a pH between 8.3 and 8.8 at room temperature, is included in standard PCRs at a concentration of 10 mM. When incubated at 72°C (the temperature commonly used for the extension phase of PCR), the pH of the reaction mixture drops by more than a full unit, producing a buffer whose pH is -7.2. Monovalent cations. Standard PCR buffer contains 50 mM KC1 and works well for amplification of segments of DNA>500 bp in length. Essential components of PCR Template DNA. Template DNA containing target sequences can be added to PCR in single- or double-stranded form. Closed circular DNA templates are amplified slightly less efficiently than linear DNAs. Although the size of the template DNA is not critical, amplification of sequences embedded in high-molecular-weight DNA (>10 kb) can be improved by digesting the template with a restriction enzyme that does not cleave within the target sequence. When working at its best, PCR requires only a single copy of a target sequence as template. However, several thousand copies of the target DNA are seeded into the reaction. In the case of mammalian genomic DNA, up to 1.0 µg of DNA is utilized per reaction, an amount that contains ~3 x 105 copies of a single-copy autosomal gene. The typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10 ng, 1ng, and 1 pg, respectively. If the nucleotide sequences at the ends of a particular DNA region are known, the intervening fragment can be amplified directly by the polymerase chain reaction (PCR) The PCR depends on the ability to alternately denature (melt) double-stranded DNA molecules and renature (anneal) complementary single strands in a controlled fashion. A typical PCR procedure begins by heat-denaturation of a DNA sample into single strands. Two synthetic oligonucleotides complementary to the 3' ends of the target DNA segment of interest are added in great excess to the denatured DNA Temperature is lowered to 50–60°C. These specific oligonucleotides, which are at a very high concentration, will hybridize with their complementary sequences in the DNA sample, whereas the long strands of the sample DNA remain apart because of their low concentration. The hybridized oligonucleotides serve as primers for DNA chain synthesis in the presence of deoxynucleotides (dNTPs) and a temperature-resistant DNA polymerase such as Taq polymerase, can remain active even after being heated to 95°C and can extend the primers at temperatures up to 72°C. When synthesis is complete, the whole mixture is then heated to 95°C to melt the newly formed DNA duplexes. After the temperature is lowered again, another cycle of synthesis takes place because excess primer is still present. Repeated cycles of melting (heating) and synthesis (cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequence between the primer sites is doubled; therefore, the desired sequence increases exponentially—about a million-fold after 20 cycles—whereas all other sequences in the original DNA sample remain unamplified. Types of PCR  Hot-Start PCR  Touch Down  Long and Accurate PCR (LA)  Inverse PCR  Nested PCR  Real Time PCR Real Time PCR Real time or quantitative PCR (sometimes called kinetic PCR) is used in research laboratories to quantify gene expression and to confirm differential expression of genes detected by array technology. In analytical laboratories, real time PCR is used to measure the abundance of particular DNA or RNA sequences in clinical and industrial samples and, in both types of laboratories, to screen for mutations and single nucleotide polymorphisms. Real time PCR uses commercially available fluorescence-detecting thermocyclers to amplify specific nucleic acid sequences and measure their concentration simultaneously. Because the target sequences are amplified and detected in the same instrument, there is no need to withdraw aliquots during the reaction or to process them. The instrument plots the rate of accumulation of amplified DNA over the course of an entire PCR. The greater the initial concentration of target sequences in the reaction mixture, the fewer the number of cycles required to achieve a particular yield of amplified product. The initial concentration of target sequences can therefore be expressed as the cycle number (Cycle threshold CT) required to achieve a present threshold of amplification. A plot of CT against the log10 of the initial copy number of a set of standard DNAs yields a straight line. The target sequences in an unknown sample may be easily quantified by interpolation into this standard curve. What’s Wrong With Agarose Gel Detection? Low sensitivity Low resolution Non-automated Size-based discrimination only Results are not expressed as numbers (based on personal evaluation) Ethidium bromide staining is not very quantitative End point analysis In real-time PCR, the amount of DNA is measured after each cycle by the use of fluorescent markers that are incorporated into the PCR product. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision. Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction. Amplification plot Real time PCR chemistries DNA binding dyes: SYBR Green Probe based chemistries: Taqman Probes, Molecular beacon probes, Scorpion Probes Quenched dye primers: Amplifluor primers, LUX primers DNA-Binding Dyes (SYBR Green I) It is an inter-chelating , asymmetric cyanine dye The most commonly used DNA-binding dye for real-time PCR is SYBR Green I, which binds non specifically to double-stranded DNA (dsDNA). SYBR Green I exhibits little fluorescence when it is free in solution, but its fluorescence increases up to 1,000- fold when it binds dsDNA Therefore, the overall fluorescent signal from a reaction is proportional to the amount of dsDNA present, and will increase as the target is amplified. 494 absorbance - 521 emission 3’ 5’ 5’ 3’ PCR makes more double-stranded DNA 3’ Taq 5’ 5’ 3’ Taq SYBR Green dye binds to dsDNA l l l 3’ Taq5’ 3’ Taq l l When illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm. The advantages of using dsDNA-binding dyes Simple assay design (only two primers are needed; probe design is not necessary) Ability to test multiple genes quickly without designing multiple probes (e.g., for validation of gene expression data from many genes in a microarray experiment) Lower initial cost (probes cost more) The ability to perform a melt-curve analysis to check the specificity of the amplification reaction. The major drawbacks of DNA-binding dyes Lack of specificity, that is, DNA-binding dyes bind to any dsDNA. As a result, the presence of nonspecific products in a real-time PCR reaction may contribute to the overall fluorescence and affect the accuracy of quantification. Another consequence is that DNA-binding dyes cannot be used for multiplex reactions because fluorescent signals from different amplicons cannot be distinguished. Instead, you can set up parallel reactions to examine multiple genes, such as a gene of interest and reference gene, in a real-time PCR assay with SYBR Green I. Taq DNA Polymerase 1 has 5’ to 3’ DNA polymerase activity 5’ to 3’ DNA exonuclease activity & lacks 3’ to 5’ DNA exonuclease [proof reading] activity Probe based chemistries-Taqman Probes Assay Components consist of a pair of unlabeled primers and A labelled TaqMan probe TaqMan probes are labeled with two fluorescent dyes that emit at different wavelengths The probe is intended to hybridize specifically in the DNA target region of interest between the two PCR primers. The “reporter” (R) dye is attached at the 5′-end of the probe sequence while the “quencher” (Q) dye is synthesized on the 3′-end. When the probe is intact and the reporter dye is in close proximity to the quencher dye, little to no fluorescence will result because of suppression of the reporter fluorescence due to an energy transfer between the two dyes. During polymerization, strand synthesis will begin to displace any TaqMan probes that have hybridized to the target sequence. The Taq DNA polymerase used has a 5′-exonuclease activity and therefore will begin to chew away at any sequences in its path (i.e., those probes that have annealed to the target sequence). When the reporter dye molecule is released from the probe and is no longer in close proximity to the quencher dye, it can begin to fluoresce Increase in the fluorescent signal results if the target sequence is complementary to the TaqMan probe. Reporters- Texas red, cyanine 3, tetrachlorofluorescein (TET), tetra methyl rhodamine (TAMRA) Quenchers: Black Hole Quenchers, TAMRA Disadvantages- Design a probe for every gene. HOT START PCR Mis-pairing of primers which occurs at suboptimal annealing temperatures leads to non specific PCR products. Hot start PCR is a method to optimize the yield of the desired amplified product in polymerase chain reactions and to suppress nonspecific amplification. This is done by withholding an essential component of the PCR — the DNA polymerase or Mg2+ or primer, for example — until the reaction mixture has been heated to a temperature that inhibits hybridization of primers to one another or to non specific regions of the template. Hot start PCR is not essential in optimized simple PCRs that contain a single pair of well-designed primers and generate high yields of a specific amplified product. However, the method is important when nonspecific amplification is a problem — for example, when fewer than 104 copies of template DNA are present in the reaction, when the template DNA is highly complex (e.g., mammalian genomic DNA), or when PCRs contain several pairs of oligonucleotides (multiplex PCR) Initially in hot start PCR template DNA and primers were mixed together and held at a temperature above the threshold of nonspecific binding of primer to template. All of the components necessary for the extension phase of the PCR were then added except the thermostable polymerase. Thermal cycling was initiated after the final addition of the DNA polymerase to the preheated reaction mixture. The elimination of the warm-up phase preceding the first cycle of PCR reduced the opportunities for nonspecific annealing of oligonucleotide primers, whereas the absence of DNA polymerase activity prevented extension of mismatched primers. This original method was difficult because the reaction mixtures were assembled in tubes whose temperature was maintained by a heating.  MODIFIED METHODS OF HOT START PCR Variation 1.The most popular method involves creating a physical barrier of wax between components of the reaction. For example, the primers, Mg2+, dNTPs, and buffer are mixed at room temperature in the bottom of the reaction tube and then covered with melted wax (e.g., Ampliwax PCR Gems from Perkin-Elmer or a commercial wax that melts at low temperature [melting point = 53-55°C]). The wax solidifies on cooling and confines the reagents to the bottom of the tube. The remaining components of the reaction are then mixed on top of the wax barrier. During the denaturation step of the first cycle of PCR, the wax barrier melts, allowing the components of the reaction to merge. The melted wax floats to the top of the reaction mixture where it now acts as a barrier to evaporation. Variation 2. Hot start PCR includes the use of neutralizing monoclonal antibodies to inhibit polymerase activity during the assembly and warm-up phase of the reactions. When the temperature increases, the antibody dissociates from the enzyme and is inactivated during the denaturation step of the first cycle of PCR. Variation 3. Mg2+ embedded in wax beads (HotWax Beads) is released into the reaction mixture as the wax melts. HotWax Beads are sold in several formulations that provide predetermined concentrations of Mg2+ for PCRs of standard volume. Touch Down PCR Touch Down PCR increases the specificity of PCR by using higher annealing temperatures at the earlier cycles and increases the efficiency by lowering the annealing temperatures gradually The principle behind this is that the annealing temperature during a PCR reaction determines the specificity of primer annealing. At high annealing temperatures, only very specific base pairing between the primer and the template will occur and thus the first sequence amplified is the one between the regions of greatest primer specificity and the one of interest. Then the amplified fragments will further be amplified during subsequent runs at lower temperatures with more efficiency. In this way, the number of amplified targeted sequence would be in excess compared to the number of amplified non-specific sequences. This method dramatically increases the quality of outcome of PCR. A typical Touch Down PCR cycling condition has two phases. The first phase of touchdown programming uses an annealing temperature that is approximately 10°C above the calculated Tm (melting temperature). The temperature is reduced by 1°C every successive cycle until the calculated Tm range is reached. This is done for a total of 10-15 cycles. Phase 2 follows generic PCR amplification of up to 20-25 cycles using the final annealing temperature reached in the touchdown phase. The cycles and temperature drop during touchdown phase can be adjusted from 1-3 cycles per 1-3°C drop in temperature if non-specific products are still observed or if the yield is low Long and accurate PCR Under standard reaction conditions, PCR amplifies segments of DNA 1-2 kb in length. This capacity is sufficient for many routine manipulations of DNA (e.g., sequencing and mutagenesis), but it is not enough to amplify an entire mammalian gene — even one of modest size — nor a cDNA of average dimensions. Explanations proposed for the inability of standard PCR to amplify long segments of DNA include: Damage to the template and product DNAs during exposure to high temperature in buffers that may not adequately maintain control over pH. The presence of stray divalent cations (Mn2+ is always the prime suspect) that may promote cleavage of DNA at high temperature. Difficulties in denaturing very long DNA molecules during the heating step of the PCR cycle. The inability of DNA polymerases to remain attached to the template DNA, particularly in regions of secondary structure. The high rate of incorporation of incorrect bases by thermo stable polymerases such as Taq that lack an editing function. The incorporation of a mismatched base at the 3' end of a growing strand may cause the enzyme to seize up and may limit the size of the PCR product. Note DNA Polymerases can be Distributive or Processive Long and accurate PCR Long and accurate polymerase chain reaction (LA PCR) refers to the production of amplified product longer than ∼3 kilobases (kb) with high fidelity. Long PCR mixtures typically yield PCR products with some tenfold fewer mutations than those observed in products resulting from conventional PCR. The obstacles created by mismatched 3' termini could be eliminated by using two different thermostable DNA polymerases to catalyze the amplification reaction. One of the polymerases would be an efficient but error-prone workhorse, whereas the second, used in much smaller amounts, would provide a 3'—>5' exonuclease function that would resect mismatched ends. The mixture of DNA polymerases typically included either Taq or Klentaq1 (which have no 3′- exonuclease proofreading activity) as the major component and, as the minor component, an archaebacterial DNA polymerase (with proofreading activity) such as Deep Vent, Vent or Pfu1. Since the introduction of mixtures of DNA polymerases, most PCRs of any length have improved in reliability and in yield of product. Nested PCR Nested PCR is used to increase the specificity of DNA amplification by reducing the non-specific amplification of DNA. A nested PCR assay has 2 sets of primers (outer pair and inner pair) for a single locus and two successive PCRs. In the first PCR run, the outer pair primers are used to generate DNA products as the regular PCR does and thus their DNA products may contain non-specifically amplified DNA fragments. These products then enter a second run of PCR using the second set “inner” primers whose binding sites are completely or partially different from and located after the 3’end of each of the outer pair primer used in the first PCR reaction. Therefore a second PCR product was produced and shorter than the first one. If the wrong locus was amplified by mistake in the first round, it’s very unlikely it would also be amplified a second time by the second pair of primers, thus in this way nested PCR greatly increases the specificity of PCR. Nested primers are used as important control for many experiments with unknown genome sequence. For example, when amplifying a particular member of a polymorphic gene family or amplifying from a clinical specimen containing a heterogeneous population of sample input. A drawback with this technique is that addition of a second pair of primers after the first PCR run increases the chance of nonspecific contamination. Inverse PCR Standard PCR amplifies segments of DNA that lie between two inward-pointing primers. By contrast, inverse (also known as inverted or inside-out) PCR is used to amplify unknown DNA that flanks one end of a known DNA sequence and for which no primers are available. The technique was developed independently by several groups (Ochman et al. 1988; Triglia et al. 1988; Silver and Keerikatte 1989). Inverse PCR involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intra-molecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences. Both upsteam and downstream flanking regions can be obtained in a single inverse PCR by using a restriction enzyme that does not cleave within the known DNA sequence. Applications: Generate end-specific probes for chromosome walking Clone unknown cDNA sequences from total RNA Recover integration sites used by viruses, transgenes, and transposons Inverse PCR