Fragments of DNA can be produced by several methods, including: conversion…

The idea

There are three routes to a DNA fragment. First, complementary DNA: choose a cell that makes the protein in quantity, so it is rich in the relevant mRNA - beta cells of the pancreas for insulin - extract the mature mRNA, and use reverse transcriptase with free DNA nucleotides to build a single-stranded DNA copy; DNA polymerase then adds the second strand. Because the mRNA has already been spliced, cDNA contains only exons, so a bacterium can express it. Second, restriction endonucleases: each enzyme recognises one specific palindromic sequence and hydrolyses phosphodiester bonds in the sugar-phosphate backbone there. A staggered cut leaves sticky ends, short single-stranded runs of exposed bases that can later pair with complementary ends. Third, the gene machine: if the base sequence is known, the gene can be synthesised from free nucleotides, with no introns and no need for mRNA.

What earns the marks

• Using reverse transcriptase with free DNA nucleotides to make cDNA from mRNA.
• Explaining that cDNA contains no introns because the mRNA has already been spliced.
• Restriction endonucleases cutting the DNA at specific base sequences, that is at recognition or restriction sites, accepting palindromic sequences.
• A staggered cut producing sticky ends, short exposed single-stranded sections of bases.
• The gene machine building the gene from a known base sequence, free of introns.

Common mistakes and how to put them right

• Saying reverse transcriptase makes mRNA from DNA, which is the wrong direction.

• Saying restriction enzymes cut at random, or that they simply 'cut the gene out', with no recognition sequence named.

• Describing sticky ends as complementary amino acids or as complementary genes.

• Choosing any cell to extract mRNA from, when the mark is for a cell that actively produces the protein.

• Saying the restriction enzyme breaks the hydrogen bonds between the strands, when it hydrolyses phosphodiester bonds in the backbone.

Exam tips

• If a question gives you a choice of method, cDNA is the safe answer for a eukaryotic gene because of the introns.
• Use the phrase 'specific base sequence' or 'recognition site' whenever restriction enzymes appear - it is where the mark sits.
• Say which bond is broken or formed; the examiner is checking you know phosphodiester bonds from hydrogen bonds.