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 clectrophoresis, 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.

Short answer

Question: Determine the position of restriction-enzyme cutting sites and the distance between them in a cloned DNA segment of 1300 bp in length, using the given fragment sizes produced by enzyme I, enzyme II, and their combination.

Step-by-step solution

Understand the data given

We are given the number and size of fragments produced by enzymes I, II, and their combination after a gel electrophoresis. We need to use this data to map the restriction sites and the distance between them.

Enzyme I Fragment Analysis

Analyze the fragments produced by enzyme I. Observe the fragment sizes and determine the possible positions of enzyme I cutting sites.

Enzyme II Fragment Analysis

Analyze the fragments produced by enzyme II. Observe the fragment sizes and determine the possible positions of enzyme II cutting sites.

Combined Enzymes Fragment Analysis

Analyze the fragments produced by the combined action of enzymes I and II. Observe the fragment sizes and use this information to deduce the sequence of enzyme cutting sites on the DNA segment.

Construct the Restriction Map

Use the information about the cutting sites and fragment sizes obtained from the analyses in steps 2-4 to construct a restriction map. This map should show the positions of restriction-enzyme cutting sites and the distance between them in base pairs.

Key concepts

Restriction Enzymes

Restriction enzymes, often referred to as 'molecular scissors,' play a crucial role in genetic engineering. These naturally occurring enzymes can identify specific sequences of DNA and cut the strands at or near these sequences. Each restriction enzyme has its own specific recognition sequence, which typically consists of four to eight base pairs. The sites where these enzymes cut can either produce 'blunt' or 'sticky' ends, the latter having overhanging sequences which can be useful in DNA cloning.

When constructing a restriction map, the use of restriction enzymes is essential. You'll first digest the DNA with different restriction enzymes separately and in combination, and then compare the size of the resulting fragments to deduce where each enzyme is making its cuts. In the exercise provided, enzyme I and enzyme II recognize different sequences within the 1300 bp DNA segment, and the manner in which they cut the DNA helps determine the location of their restriction sites.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. DNA fragments are negatively charged due to their phosphate backbone, and they will move towards the positive electrode when an electric current is passed through the gel. Smaller fragments move faster and thus travel further than larger ones within the gel matrix.

After the DNA is cut by restriction enzymes, gel electrophoresis allows for the visualization of the fragments. By comparing the distances traveled by DNA fragments to a standard ruler of known lengths, you can determine the size of each fragment in base pairs. This information is crucial when constructing a restriction map as it lets us infer the positions of the cuts made by the restriction enzymes on the DNA segment.

DNA Cloning

DNA cloning is a technique used to create multiple, identical copies of a particular DNA segment. This process usually involves inserting a DNA fragment of interest into a vector, such as a plasmid, which is then introduced into a host organism like bacteria. The host organism replicates, passing along the inserted DNA to its progeny, thereby amplifying the desired DNA segment.

In the context of our exercise, the cloned DNA segment refers to the piece of DNA that has been copied multiple times within a host organism. This amplification makes it easier to study the DNA in detail, such as identifying its nucleotide sequence or analyzing its function. For creating a restriction map, cloning ensures that there is enough DNA to perform multiple digestion and analysis processes.

Base Pairs

Base pairs are the building blocks of the DNA double helix. In DNA, there are four types of nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C). Adenine pairs with thymine, and guanine pairs with cytosine. The sequence of these base pairs determines the genetic information carried by a DNA molecule.

Knowing the number of base pairs that compose a DNA fragment is essential in various genetic techniques, including restriction mapping. In the restriction map construction exercise, the total length of the DNA segment, 1300 base pairs, gives a framework within which the sizes of the fragments produced by the restriction enzymes can be interpreted. By sorting these fragments according to their base pair lengths as determined by gel electrophoresis, you can deduce the order of restriction sites and thus create a map detailing the distances between the sites on the DNA molecule.