(Image from www.rcsb.org/pdb/molecules/ pdb8_3.html
Protein Data Bank, 2003)

PURPOSE

Restriction endonucleases, or restriction enzymes, are enzymes that aid in the digestion of double-stranded DNA. They are like highly specialized scissors that can only cut within their own DNA recognition sequence. Recognition sequences typically consist of 4 to 6 DNA nucleotides in palindrome form, so that the two strands of the double helix have the same sequence when read in opposite directions (unlike our 26-letter alphabet, the DNA alphabet consists of only 4 nucleotide letters: G, A, T, and C). Nowadays, more than 3500 restriction enzymes have been identified. About 500 have been commercialized, making them an extremely versatile and accessible tool in our molecular biology toolbox.

In this activity we will use restriction enzymes to analyze a double-stranded, circular form of PhiX174 DNA (called RF DNA for Replicative Form). Since all of its 5386 base pairs (commonly abbreviated as 5386 bp or 5.39 kb) have been sequenced, we can predict exactly which DNA fragments should be produced after digestion with certain restriction enzymes. With the help of a free web-based tool called NEBcutter, we can also predict what will happen when we load these DNA fragments onto an agarose gel for separation by gel electrophoresis. Pores in the agarose gel are barely big enough to let the DNA fragments pass through. When a mixture of differently sized DNA fragments are applied to one side of the gel, and a current is applied across the gel, negatively charged DNA fragments will start to travel across the gel towards the positive charge. Since larger fragments will have a harder time squeezing their way through the agarose pores, they will lag behind the smaller fragments in a predictable way.

(Image from www.rtc.riken.go.jp/jouhou/image/dna-protein/all/all.html, 2003)

KEY CONCEPTS

After this activity, students will be able to:

• define restriction endonuclease, DNA recognition sequence, palindrome, restriction digest, gel electrophoresis, DNA fragment, base pairs/kb, restriction map
• use the NEBcutter website to prepare a restriction map
• perform restriction digests and run an agarose gel
• interpret the banding pattern on a gel and relate it to the restriction map
• understand the use of controls
• prepare and use a standard curve to determine DNA fragment length (optional)