Home -> Step by step guides -> Forensic amplification of dna (PCR)
Necessary Equipment and Software:
- Thermocycler: A machine that heats and cools the reaction tubes to the precise temperatures needed for each step of the process.
- PCR tubes: Special thin-walled tubes that allow for rapid thermal changes.
- Micropipettes: Used for accurate measurement and transfer of solutions.
- Vortex mixer: To mix reagents evenly.
- Centrifuge: For spinning down your DNA template.
- DNA Template: The DNA that contains the sequence you want to amplify.
- Primers: Short pieces of DNA that are complementary to the sequences on either side of the section of DNA you want to copy.
- Deoxynucleotide triphosphates (dNTPs): The building blocks for new DNA strands.
- Taq Polymerase: An enzyme that synthesizes the new DNA strands.
- Buffer solution: To create an optimal environment for the Taq polymerase.
- DNA loading dye: Used in DNA visualization in the gel electrophoresis step.
Step 1: Preparation of the Reaction Mixture
Involves mixing together your DNA template, primers, Taq polymerase, dNTPs, and buffer solution in a PCR tube. The ratio and volume of these elements would depend on the specific guidelines of the Taq polymerase that is being used.
Step 2: Programming the Thermocycler
Thermocycler must be programmed to change temperatures at the correct times. A typical program looks like this:
- Denaturation: 94-96°C for 1 minute.
- Annealing: 50-65°C (depends on primers used) for 1 minute.
- Extension: 72°C for 1 minute.
These steps are repeated for about 30 cycles.
Step 3: Denaturation
The PCR tube is heated to about 95°C. This high temperature breaks the hydrogen bonds between the bases of the DNA strands, causing the double-stranded DNA to denature into two single strands.
Step 4: Annealing
The temperature is quickly reduced to about 55°C which encourages the primers to bond to their complementary sequences on the single-stranded DNA templates.
Step 5: Extension (Elongation)
The temperature is raised to about 72°C. This is the optimum temperature for the Taq polymerase to add dNTPs onto the end of the primer, synthesizing a new strand of DNA.
Step 6: Final Elongation
After the cycles, the reaction mixture is kept at 72°C for about 5-10 minutes to ensure that any remaining single-stranded DNA is fully replicated.
Step 7: Hold
The thermocycler is set to 4°C for short-term storage of the PCR reaction or 20°C for longer-term storage.
Step 8: Gel Electrophoresis
This step is performed if you wish to visualize the amplified DNA. The PCR product is mixed with a loading dye and loaded into a gel. A current is applied across the gel, moving the negatively-charged DNA towards the positive end. The smaller fragments move faster and end up further away from the starting well.
PCR basics and principles:
PCR is based on the natural processes a cell uses to replicate a new DNA strand. This method uses cycles of repeated heating and cooling to achieve the denaturation, annealing, and extension of the DNA strands. The foundational concept of PCR is to generate thousands to millions of copies of a specific DNA sequence from a DNA template. This is achieved by using the enzymes and substrates needed by a cell to replicate DNA, but in an in vitro (test tube) setting. The most important foundational elements are the DNA polymerase and the primers, as they are responsible for the strand synthesis and deciding the exact section of DNA to be amplified, respectively.