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Covers the principles of Polymerase Chain Reaction (PCR) and the separation of DNA fragments via gel electrophoresis.
How can a single microscopic droplet of blood at a crime scene provide enough genetic material to identify a suspect with near-certainty?
The Polymerase Chain Reaction (PCR) is a technique used to 'amplify' or create millions of copies of a specific DNA segment. This process occurs in a thermal cycler and relies on a heat-stable enzyme called Taq polymerase, originally found in hot-spring bacteria. Each cycle consists of three temperature-dependent steps. First, Denaturation () breaks the hydrogen bonds between the two DNA strands. Second, Annealing () allows short DNA sequences called primers to bind to the target sequence. Finally, Extension () is when Taq polymerase adds nucleotides to the primers, synthesizing new DNA strands. Because the DNA doubles every cycle, the growth is exponential.
Quick Check
Why is it necessary to use Taq polymerase instead of human DNA polymerase in a PCR reaction?
Answer
Human DNA polymerase would denature (unfold and lose function) at the high temperatures () required for the denaturation step, whereas Taq polymerase is heat-stable.
If you start with a single double-stranded DNA molecule, how many molecules will you have after 4 cycles of PCR?
1. Identify the starting amount: . 2. Identify the number of cycles: . 3. Apply the formula: . 4. Calculate: molecules.
A researcher starts with 50 copies of a specific gene. After 20 cycles of PCR, how many copies are theoretically present?
1. 2. 3. 4. Since , then . 5. copies.
Once DNA is amplified, we use Gel Electrophoresis to sort the fragments by size. DNA has a negative charge due to its phosphate backbone. When placed in an agarose gel and subjected to an electric field, DNA migrates toward the positive electrode (anode). The gel acts like a molecular sieve: smaller fragments move through the pores faster and travel further than larger fragments. To determine the exact size of these fragments, we run a molecular ladder (a mixture of DNA fragments of known sizes) in a parallel lane for comparison.
Quick Check
In a gel electrophoresis run, which fragment will be found closest to the starting wells: a 200 base pair (bp) fragment or a 1000 bp fragment?
Answer
The 1000 bp fragment, because larger fragments move more slowly through the gel matrix.
You are analyzing a gel. The molecular ladder has bands at , , and . Your sample shows a single band that has traveled further than the marker but not as far as the marker.
1. Recall: Distance traveled is inversely proportional to size. 2. Since it traveled further than 1000 bp, it must be smaller than 1000 bp. 3. Since it traveled less than 500 bp, it must be larger than 500 bp. 4. Conclusion: The fragment size is between and .
During which stage of PCR do the primers attach to the DNA template?
If you start with 2 molecules of DNA, how many will you have after 3 cycles?
In gel electrophoresis, DNA fragments migrate toward the negative electrode because DNA is positively charged.
Review Tomorrow
In 24 hours, try to sketch the three stages of PCR and write down the formula for calculating DNA yield from memory.
Practice Activity
Look up a real image of a 'DNA ladder' online and try to estimate the sizes of the bands in the experimental lanes next to it.