Question

You have recovered a cloned DNA segment from a vector and determine that the insert is 1300 bp in length. To characterize this cloned segment, you isolate the insert and decide to construct a restriction map. Using enzyme I and enzyme II, followed by gel electrophoresis, you determine the number and size of the fragments produced by enzymes I and II alone and in combination, as recorded in the following table. Construct a restriction map from these data, showing the positions of the restriction-enzyme cutting sites relative to one another and the distance between them in units of base pairs. \(\begin{array}{cc}\text { Enzyme } & \text { Restriction Fragment Sizes (bp) } \\\ \text { I } & 350,950 \\ \text { II } & 200,1100 \\ \text { I and II } & 150,200,950\end{array}\)

Short answer

Question: Using the information provided, determine the positions and distances of the restriction enzyme cutting sites within the DNA segment and construct a restriction map. Answer: The restriction map can be represented as follows: - Enzyme I: |----350 bp----| (cut) - Enzyme II: |----200 bp----| (cut) - Enzyme I: ------------------------|----950 bp----| (cut)

Step-by-step solution

Analyze the given data

We have the following fragment sizes for each enzyme: - Enzyme I: 350, 950 - Enzyme II: 200, 1100 - Enzymes I and II (combined): 150, 200, 950

Determine the possible orientations of the fragments

Consider the fragments generated by enzyme I (350 and 950). Since the total length of the DNA segment is 1300 bp, it means that enzyme I cut at 350 bp from one end and 950 bp from the other end. The same applies to enzyme II, which cut at 200 bp and 1100 bp from the two ends. Next, let's look at the fragments generated by the combination of enzyme I and II. We have three fragments of sizes 150, 200, and 950 bp. Since we already know that enzyme I produces a 950 bp fragment, it suggests that enzyme II cuts within that 950 bp fragment, producing two smaller fragments of sizes 150 bp and 200 bp.

Construct the restriction map

Now we can determine the positions of the restriction enzyme cutting sites relative to one another. We know that enzyme II cuts the 950 bp fragment at 200 bp from one end and 150 bp from the other end. Therefore, the first Enzyme I cutting site is 350 bp from one end, followed by the enzyme II cutting site at 200 bp, and finally, the second Enzyme I cutting site at 950 bp (measured from the start of the segment), yielding a total of 1300 bp. So the restriction map can be represented as follows: Enzyme I: |----350 bp----| (cut) Enzyme II: |----200 bp----| (cut) Enzyme I: ------------------------|----950 bp----| (cut) These distances and cutting sites describe the restriction map of the cloned segment.

Key concepts

Molecular Genetics

Molecular genetics is a fundamental aspect of biology focusing on the structure and function of genes at a molecular level. It involves the analysis of DNA sequences to understand genetic variation and the mechanisms of genetic inheritance.

Molecular genetics techniques, such as DNA cloning, are used to manipulate genes to study their effects on organisms. The concept is critical in research and has applications in medicine, biotechnology, and genetics counseling, among other fields. By constructing and analyzing restriction maps, scientists can examine the genetic blueprint of organisms which aids in identifying gene locations, mutations, and functions.

DNA Cloning

DNA cloning is a method used to make exact copies of a specific DNA segment. This process begins by inserting a DNA fragment into a small piece of DNA known as a vector, which can replicate within a host cell. Once inside the host, the DNA segment is duplicated along with the vector's replication, creating multiple copies of the DNA of interest.

Cloning allows for the amplification and isolation of particular DNA regions for further study, and it's an essential step for restriction mapping, where cloned segments are analyzed using restriction enzymes to determine genetic sequences and map out genetic markers.

Gel Electrophoresis

Gel electrophoresis is a technique widely used to separate DNA fragments based on their size. During the process, a current is passed through a gel matrix where the DNA samples have been loaded. DNA molecules are negatively charged due to their phosphate backbone, so they migrate towards the positive electrode. Smaller fragments move through the gel faster than larger ones, resulting in bands that can be visualized and measured.

This technique is critical in the field of molecular biology for analyzing DNA after it has been cut by restriction enzymes, helping to construct a map which reveals the location of various restriction sites within the DNA - a process central to the exercise at hand.

Restriction Enzymes

Restriction enzymes are proteins that cut DNA at specific sequences, producing predictable fragments. These enzymes are pivotal in molecular biology for DNA modification and mapping. They recognize short, specific sequences within the DNA molecule and cleave the phosphodiester bond within or near those sequences.

During a restriction digest, as performed in the provided exercise, these enzymes act as molecular scissors to cut the DNA at known sites. The resulting fragments can be sorted by size using gel electrophoresis. By comparing the size of these fragments with the action of different restriction enzymes, researchers can deduce the locations of the cutting sites and construct detailed restriction maps, ultimately leading to a greater understanding of the DNA's structure and function.