(e) the principles and uses of electrophoresis for separating nucleic acid…

The idea

Electrophoresis separates charged molecules by size using an electric field through a gel. Nucleic acids carry negative phosphate groups, so they migrate towards the positive electrode; proteins are separated after denaturation and are usually given a uniform negative charge by SDS, so they too move towards the positive electrode and separate by mass. The gel acts as a molecular sieve: small fragments travel further and faster than large ones in a set time, producing bands. Fragment size is estimated by comparison with a standard ladder of known sizes. Uses include checking PCR products, restriction digests and plasmid digests, separating DNA fragments for sequencing or blotting, and separating proteins for analysis such as Western blotting. The principle is charge-based migration plus size-dependent sieving; the use is resolving and identifying fragments or proteins.

What earns the marks

• Nucleic acids are negatively charged because of their phosphate groups, so they move towards the positive electrode (anode) in an electric field.
• The gel acts as a molecular sieve: smaller fragments migrate further and faster than larger fragments in a given time, so separation is by size.
• Proteins are separated after denaturation and are typically given a uniform negative charge (for example by SDS), so they migrate towards the positive electrode and separate mainly by mass.
• Fragment sizes are estimated by comparison with a standard ladder of fragments of known sizes run alongside the sample.
• Uses include checking PCR products or restriction digests, resolving DNA fragments before sequencing or blotting, and separating proteins for analysis such as Western blotting.

Common mistakes and how to put them right

• Thinking DNA moves towards the negative electrode.

  Put it right: DNA is negatively charged and moves towards the positive electrode.

• Assuming separation depends only on charge.

  Put it right: In a gel the sieving effect means size also determines how far a fragment travels.

• Believing larger fragments travel furthest.

  Put it right: Smaller fragments move further and faster through the gel mesh.

• Treating electrophoresis as a way to amplify DNA.

  Put it right: It separates existing molecules; amplification is done by PCR.

Exam tips

• State the direction of movement and link it explicitly to the charge on the molecule.
• Explain the gel as a sieve so that size, not just charge, explains the band positions.
• When asked about uses, name a concrete application such as checking a restriction digest or separating proteins for blotting.