PCR – diagnostics. Aizhan Zhussupova
flanking regions are not copied into the mature mRNA, but often contain sequences which affect the formation of 3’ and 5’ ends of the message; http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.html
Figure 2.3. Diagram of molecular cloning using bacteria and plasmids. Modified from: Wikimedia Commons Kelvinsong
These enzymes can be used in the PCR to copy any nucleic acid segment of interest. Usually this is DNA; if RNA needs to be copied, it is usually first transcribed into DNA with the help of the enzyme reverse transcriptase – a method known as reverse transcription PCR (RT-PCR). For the copying procedure only a small fragment of the DNA section of interest needs to be identified. This then serves as a template for producing the primers that initiate the reaction. It is then possible to clone DNA whose sequence is unknown. This is one of the method’s major advantages.
Genes are commonly flanked by similar stretches of nucleic acids (Fig. 2.2).
Once identified, these patterns can be used to clone unknown genes – a method that has supplanted the technique of molecular cloning in which DNA fragments are tediously copied in bacteria or other host organisms (see Fig. 2.3).
With the PCR method this goal can be achieved faster, more easily and above all in vitro, i. e. in the test-tube. Moreover, known sections of long DNA molecules, e.g. of chromosomes, can be used in PCR to scout further into unknown areas. Most PCR methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size. The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses. Types in Appendix 1.
A basic PCR set up requires several components and reagents, including:
– DNA template that contains the DNA region (target) to be amplified;
– Two primers that are to the 3’ ends of each of the sense and antisense strands of the DNA target;
– Taq polymerase or another DNA-polymerase with a temperature optimum at around 70 °C;
– Deoxynucleoside triphosphates (dNTPs), the building-blocks from which the DNA polymerase synthesizes a new DNA strand;
– Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase;
– Bivalent 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.
PCR is commonly carried out in a reaction volume of 10-200 gl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction. Many modern thermal cyclers make use of the thermoelectric cooling (Peltier effect), which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. These instruments are renowned for their reliability, accuracy, and user-friendly interfaces. 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.
Figure 2.4. Various models of PCR machine. Note: from left to right: a) – «Baby Blue», a 1986 prototype machine for doing PCR; b) – an older model three-temperature thermal cycler for PCR; c) – handheld PCR machine; d) 3D Digital PCR System
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (see Fig. 2.5).
Figure 2.5. Basic stages of PCR. From: Wikimedia Commons Enzoklop
The cycling is often preceded by a single temperature step 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 (only required for DNA polymerases that require heat activation by hot-start PCR): This step consists of heating the reaction to a temperature of94-96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1-9 minutes.
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-stranded DNA molecules.
Annealing step: The reaction temperature is lowered to 5065 °C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature needs to be low enough to allow for hybridization of the primer to the strand, but high enough in order for the hybridization to be specific, i.e. the primer should only bind to a perfectly complementary part of the template.
If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind. 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 formation.
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 (this is the temperature needed for optimal activity for most polymerases used in PCR) 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 by applying an electric field to move the negatively charged molecules through the matrix of an appropriate substance. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. 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 (see Fig. 2.6).