Biotechnology: PCR, Gels & CRISPR

You’ll be able to

Explain how PCR amplifies a target DNA sequence through thermal cycling
Describe how gel electrophoresis separates DNA fragments by size and charge
Summarize how CRISPR-Cas9 is guided to edit a specific DNA sequence

PCR: copying DNA a billionfold

Polymerase chain reaction (PCR) makes millions of copies of a target DNA region in a test tube by cycling through three temperatures. Denaturation (~95°C) heats the DNA so its strands separate. Annealing (~55°C) cools it so short primers bind to the ends of the target on each strand. Extension (~72°C) lets a heat-stable Taq polymerase build new complementary strands. Each cycle doubles the number of target molecules, so the amount grows exponentially — after n cycles one molecule becomes 2ⁿ copies.

PCR amplification

copies after n cycles = starting copies × 2ⁿ

Each cycle doubles the DNA, so growth is exponential — 30 cycles turns one molecule into roughly a billion.

Gel electrophoresis: sorting by size

Gel electrophoresis separates DNA fragments by length. DNA is loaded into wells in an agarose gel and an electric field is applied. Because the phosphate backbone gives every fragment a negative charge, all fragments migrate toward the positive electrode. The gel acts like a molecular sieve: smaller fragments slip through more easily and travel farther, while larger fragments lag behind. Comparing the banding pattern to a known-size "ladder" reveals each fragment’s length — the basis of DNA fingerprinting and gene analysis (often after cutting DNA with restriction enzymes).

CRISPR-Cas9: programmable editing

Borrowed from a bacterial immune system, CRISPR-Cas9 is a precise gene-editing tool. A short guide RNA is designed to be complementary to a target DNA sequence; it leads the Cas9 enzyme to that exact site, where Cas9 acts like molecular scissors and cuts both strands of the DNA. The cell’s repair machinery then either disables the gene or, if a template is supplied, pastes in a new sequence. Because the guide RNA can be reprogrammed to any sequence, CRISPR lets researchers edit genes with unprecedented ease.

Worked example

A researcher starts a PCR reaction with a single copy of a target DNA molecule. Assuming perfect doubling each cycle, how many copies exist after 4 complete cycles?

1.Each PCR cycle doubles the number of target molecules, so use copies = starting × 2ⁿ.
2.Here the start is 1 molecule and n = 4 cycles, so copies = 1 × 2⁴.
3.Compute 2⁴ = 2 × 2 × 2 × 2 = 16.

Answer: After 4 cycles there are 2⁴ = 16 copies of the target DNA.

Checkpoint

In gel electrophoresis, DNA fragments move toward the positive electrode, and smaller fragments travel farther than larger ones. Why do the fragments move toward the positive electrode at all?

Tip

Remember the gel rule as "small and fast": shorter fragments weave through the agarose mesh more easily and end up farthest from the wells, near the positive end. Longer fragments get tangled and stay closer to where they started.

Checkpoint

In CRISPR-Cas9 gene editing, what determines which DNA sequence Cas9 will cut?

On the exam

Keep the tools straight by their jobs: PCR amplifies (makes more copies), gel electrophoresis separates and visualizes by size, and CRISPR-Cas9 edits a specific sequence. Questions often hinge on picking the right tool for the stated goal.

Answer the 2 checkpoints as you read.