Denaturation: The reaction mixture is heated to approximately . This high temperature breaks the hydrogen bonds between complementary base pairs, separating the double-stranded DNA into two single strands.
Annealing: The temperature is lowered to between and . This allows the primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates.
Elongation (Extension): The temperature is raised to , the optimum temperature for Taq polymerase. The enzyme adds free nucleotides to the primers, synthesizing a new complementary strand of DNA.
Each cycle of these three stages doubles the amount of DNA; for example, 20 cycles can produce over a million copies ().
Gel Electrophoresis is a technique used to separate DNA fragments based on their size and electrical charge by moving them through an agarose gel matrix using an electric current.
DNA molecules are negatively charged due to the phosphate groups in their backbone; therefore, when placed in an electric field, they migrate toward the positive electrode (anode).
The agarose gel acts as a molecular sieve; smaller/shorter fragments move more easily through the pores and thus travel faster and further from the starting wells than larger fragments.
To visualize the results, probes (radioactive or fluorescent) are added to bind to specific sequences, creating a visible pattern of bands known as a DNA electrophoretogram.
| Feature | PCR (In Vitro) | In Vivo Replication |
|---|---|---|
| Separation Method | Heat () | DNA Helicase enzyme |
| Polymerase Type | Taq Polymerase (Heat-stable) | DNA Polymerase (Human/Organism) |
| Primers | Synthetic DNA primers | RNA primers |
| Environment | Thermal cycler machine | Within a living cell |
Temperature Precision: Always specify the exact temperatures for PCR stages (, , ) and explain the biological reason for each (e.g., breaking H-bonds vs. enzyme optimum).
Paternity Logic: In paternity questions, remember that every band in a child's profile MUST be present in either the mother's or the biological father's profile. If a child has a band that the mother lacks, it must have come from the father.
Common Mistake: Do not confuse 'DNA Profiling' with 'DNA Sequencing'. Profiling looks at fragment lengths (VNTRs), while sequencing determines the exact order of every nucleotide base.
Verification: When analyzing a gel, check the direction of the current. DNA always moves toward the positive electrode. If the diagram shows the wells at the bottom, the fragments will move upward.
