Problem 19 - Biochemistry: Concepts and Connections

Question

The restriction endonuclease NotI recognizes the octanucleotide sequence GCGGCCGC. Calculate the expected number of \(N o t /\) cleavage sites in the bacteriophage \(\lambda\) genome, a linear DNA duplex \(48.5 \mathrm{kbp}\) in length with a \((\mathrm{G}+\mathrm{C})\) content of \(50 \%\).

Step-by-Step Solution

Answer

The expected number of NotI cleavage sites is 189.

Step 1: Understand the Sequence Recognition

The restriction enzyme NotI recognizes the specific DNA sequence "GCGGCCGC", which is an octanucleotide, meaning it is 8 bases long. NotI will cut at this exact sequence whenever it appears in the DNA.

Step 2: Calculate Probability of Sequence Occurrence

Since each base can be either G, C, A, or T, and the G+C content is 50%, assume the probability of each base being G or C is 0.5, and equally likely for A or T. For the sequence 'GCGGCCGC', which is all G's and C's, the probability of this exact sequence occurring is \((0.5)^8 = 0.00390625\).

Step 3: Calculate Number of Expected Cuts

The bacteriophage \(\lambda\) genome is \(48.5\) kilobase pairs, or \(48,500\) base pairs. Given the probability of finding the NotI sequence is \(0.00390625\), the expected number of sites is the total bases times the probability: \[48,500 \times 0.00390625 = 189.453125\].

Step 4: Round to Nearest Whole Number

Since you cannot have a fraction of a sequence site, round the expected number of cleavage sites to the nearest whole number. In this case, that would be 189 sites.

Key Concepts

Understanding Octanucleotide Sequences

An octanucleotide sequence is a string of eight nucleotides in DNA. Nucleotides are the basic building blocks of DNA, consisting of one of four possible bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The term "octanucleotide" comes from "octa-" meaning eight and "-nucleotide" referring to these DNA subunits.
NotI is a restriction enzyme that specifically recognizes and binds to the octanucleotide sequence "GCGGCCGC". The enzyme precisely detects when this eight-base sequence appears anywhere in a DNA molecule.
When NotI encounters its target sequence, it cuts or cleaves the DNA at that site. This very specific interaction is crucial for molecular biology techniques, such as cloning, where precise cutting and manipulation of DNA are required.

Bacteriophage λ Genome: Structure and Composition

Bacteriophage λ, or lambda phage, possesses a linear DNA genome elongating 48.5 kilobase pairs (kbp) in length. This refers to 48,500 base pairs that make up the viral genome. Bacteriophages, like λ, are viruses that infect bacteria specifically.
Understanding the composition of the λ genome is important in predicting cleavage site occurrence by restriction enzymes. In this exercise, the genome is noted to have a G+C content of 50%, meaning half of the bases are either guanine (G) or cytosine (C). The stoichiometric balance between G+C and the other two bases (A and T) significantly influences sequence availability for NotI's target site.
In genetic engineering, studying such bacterial viruses helps researchers understand gene function and expression, given that they act as vectors to introduce genetic material into bacterial cells.

Concept of DNA Cleavage Sites

DNA cleavage sites refer to specific locations on a DNA strand where enzymes, such as restriction endonucleases, cut the DNA. These are often within or adjacent to a recognized sequence.
The ability to identify and predict these sites is highly useful in various applications like genetic mapping, cloning, and DNA modification. For instance, the sequence "GCGGCCGC" is the site that the NotI enzyme specifically seeks and cleaves. Knowing the enzyme's "preferred" cutting site allows scientists to calculate how frequently cuts might occur in any given DNA sequence, such as the bacteriophage λ genome.
To determine this frequency, you consider both the probability of the octanucleotide sequence naturally appearing in the genome and the total size of the genome. In practical terms, understanding these dynamics is crucial for manipulating DNA in laboratory settings, facilitating advancements in genetic research.