BasicPCR

實驗概要

The  following basic protocol serves as a general guideline and a starting  point for any PCR amplification. Optimal reaction conditions (incubation  times and temperatures, concentration of Taq DNA Polymerase, primers,  MgCl₂, and template DNA) vary and need to be optimized. PCR  reactions should be assembled in a DNA-free environment. Use of "clean"  dedicated automatic pipettors and aerosol resistant barrier tips are  recommended. Always keep the control DNA and other templates to be amplified isolated from the other components. 

實驗原理

1.  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:

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

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

3) Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.

4) Deoxynucleoside  triphosphates (dNTPs; nucleotides containing triphosphate groups), the  building-blocks from which the DNA polymerase synthesizes a new DNA  strand.

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

6) 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

7) 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. 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.

2.  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. 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.

1) 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.

2) 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.

3) 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 degrees Celsius 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.

4) 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.

5) 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.

6) 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.

實驗材料

DNA

cDNA

實驗步驟

1. Add the following components to a sterile 0.5-ml microcentrifuge tube sitting on ice: 

2. Mix contents of tube and overlay with 50 μl of mineral or silicone oil.

3. Cap tubes and centrifuge briefly to collect the contents to the bottom.

4. Incubate tubes in a thermal cycler at 94°C for 3 minutes to completely denature the template. 
Perform 25–35 cycles of PCR amplification as follows:

1)        Denature 94°C for 45 s

2)        Anneal 55°C for 30 s

3)        Extend 72°C for 1 min 30 s

5. Incubate for an additional 10 min at 72°C and maintain the reaction at 4°C. The samples can be stored at –20°C until use.

6.  Analyze the amplification products by agarose gel electrophoresis and  visualize by ethidium bromide staining. Use appropriate molecular weight  standards.

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