Question

Restriction enzymes and sequences (a) Restriction enzymes are proteins that recognize specific sequences at which they cut the DNA. Two commonly used restriction enzymes are HindIII and EcoRI. Look up the recognition sequences that these enzymes each cut and make a sketch of the pattern of cutting they carry out. Consider the approximately 48,000 bp genome of lambda phage and make an estimate of the lengths of the fragments that you would get if the DNA is cut with both the HindIII and EcoRI restriction enzymes. There is a precise mathematical way to do this and it depends upon the length of the recognition sequence-a 5 cutter will have shorter fragments than an 8 cutter-explain that. (b) Find the actual fragment lengths obtained in the lambda genome using these restriction enzymes by going to the New England Biolabs website (www.neb.com) and looking up the tables identifying the sites on the lambda genome that are cut by these different enzymes. How do these cutting patterns compare with your results from (a)? (c) Plot the number of cuts in the lambda genome as a function of the length of the recognition sequence of several commercially available type II restriction enzymes. You can download the list of type II restriction enzymes from the book's website. Combine this plot with a curve showing your theoretical expectation.

Short answer

The expected fragments lengths for HindIII and EcoRI restriction enzymes were calculated based on the genome length and recognition sequences. These estimates were then validated via www.neb.com. The total number of cuts in the lambda genome for given lengths of recognition sequences of several type II restriction enzymes was plotted and compared with a theoretical expectation curve. The outcome demonstrates the impact of recognition sequence length on the fragmentation of the genome by restriction enzymes.

Step-by-step solution

Identify Recognition Sequences

Look up and identify the recognition sequences for HindIII and EcoRI restriction enzymes. HindIII cuts the DNA at the recognition sequence 'AAGCTT' and EcoRI cuts the DNA at the sequence 'GAATTC'.

Sketch Cutting Patterns

Create sketches for both enzymes showing how and where they would cut the lambda phage genome. Be sure to include the sequences you identified earlier.

Estimate Fragment Lengths

To estimate fragment lengths you need to consider that HindIII (6 nucleotides long) will cut the genome every 4^6 and EcoRI (6 nucleotides long) will cut it every 4^6 (considering that there are 4 base pairs, 2 of which are present in each of the DNA strands, and we have both strands to account for these enzymes). If a rough estimation is needed, it can be done by dividing the total length of the genome by the expected cut frequency.

Validate Estimates

Go to www.neb.com and cross-verify the cutting sites and resulting fragment lengths for the HindIII and EcoRI enzymes by looking at the tables identifying the lambda genome sites.

Interpret Comparisons

The fragment lengths defined in previous steps should be compared with the actual fragment lengths obtained by using the enzymes on the lambda genome. Discuss whether the results obtained in the mathematical model align with the practical outcomes in the website.

Plotting Number of Cuts

Using the list of type II restriction enzymes, create a plot of the number of cuts by such a restriction enzyme in the lambda genome based on the length of their recognition sequence. Label this chart clearly to show the number of cuts and the length of the recognition sequence.

Theoretical Expectation Curve

Next, a curve showing the theoretical expectation of cuts in the lambda genome will be created. This curve should represent the expected number of cuts for a given length of the recognition sequence. Combine this chart with previous steps.

Compare Plots

Finally, compare the initial plot with the theoretical expectation curve. Discuss any similarities or differences, and consider potential reasons for these differences.

Key concepts

Recognition Sequences

Restriction enzymes, also known as restriction endonucleases, are proteins that have the unique ability to recognize specific sequences of nucleotides in DNA, known as recognition sequences. These sequences are typically palindromic, meaning they read the same forwards and backwards on the complementary strands. Different restriction enzymes have unique recognition sequences, which they use to identify where to cut the DNA strand. For instance:

• HindIII recognizes the sequence 'AAGCTT'.
• EcoRI recognizes the sequence 'GAATTC'.

This specificity forms the basis for using restriction enzymes in DNA manipulation and cloning. Recognition sequences usually consist of 4 to 8 nucleotides, and the number of nucleotides determines the frequency of cuts along a DNA sequence. This is because every additional nucleotide reduces the probability of that sequence occurring by a factor of four, given the four nucleotides that DNA consists of.

DNA Fragmentation

DNA fragmentation is the process of cutting DNA into smaller pieces using restriction enzymes. Each enzyme cleaves the DNA at its specific recognition sequence, resulting in a pattern of fragments. The lambda phage genome, which is about 48,000 base pairs in length, can be used as a model to see how DNA fragmentation works. The length of the fragments for each enzyme can be estimated based on the sequence it recognizes and cuts.

• For a 6-base pair recognition sequence, such as those for HindIII and EcoRI, the enzyme cuts, on average, every 4⁶ base pairs (about every 4096 base pairs).
• This results in approximately 48,000/4096, or about 12 fragments, assuming random distribution of the recognition sequences along the genome.

However, the actual number and size of fragments depend on the exact locations of the recognition sequences in the genome.

Lambda Phage Genome

The lambda phage is a virus that infects bacteria, and its genome is frequently used in molecular biology experiments due to its manageable size and well-studied sequence. Its genome is approximately 48,000 base pairs long, making it an ideal candidate for studying DNA fragmentation with restriction enzymes.

• The sequence of the lambda phage genome is fully mapped, making it easy to plan and predict how restriction enzymes will cut it.
• This prediction is important in both theoretical exercises and practical applications, such as cloning or genomic mapping.

Using databases like the one provided by New England Biolabs can offer clarity by specifying the actual cut patterns of various restriction enzymes across the lambda phage genome.

Mathematical Estimation

Mathematical estimation in the context of restriction enzymes involves predicting the number and sizes of DNA fragments created when the DNA is digested with a restriction enzyme. This estimation is a powerful tool in genomics because it can predict experimental outcomes.

• The formula for determining the expected fragment size is based on the probability of finding the recognition sequence in a sequence of random nucleotides, calculated as 4ⁿ, where n is the length of the recognition sequence.
• This helps in estimating the average number of cuts and thereby the average size of the resulting fragments.

For example, a 6-base pair recognition sequence will statistically appear once every 4096 base pairs in a random sequence, providing an estimation basis for expected fragment sizes when analyzing complex genomes like that of the lambda phage.