Restriction Enzyme Crime Lab (David Gisis)

Title:

• DNA Fingerprinting Using Restriction Enzymes

Principle(s) Investigated:

• DNA editing
• Endonuclease enzyme activity
• DNA identification via RFLP analysis

Standards:

• HS-LS1-1 From Molecules to Organisms: Structures and Processes: Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.
• HS-LS3-3 Heredity: Inheritance and Variation of Traits: Apply concepts of statistics and probability to explain the variation and distribution of expressed traits in a population.

Materials:

• Restriction Enzyme activity sheet (available here)
• Pencil/Pen

Procedure:

1. distribute activity sheets (students may work individually or in pairs)
2. search suspects' DNA sequences and identify ApaLI cut sites
3. mark specific cut location on all DNA sequences
4. count basepairs in each fragment and input information into chart
5. compare restriction fragment lengths to identify matching DNA sequences

Student prior knowledge:

• familiarity with structure and functions of DNA
• familiarity with DNA replication processes and necessary enzymes

Explanation:

Restriction Fragment Length Polymorphism (RFLP) analysis was the original genetic analysis tool and has found multiple applications in medicine, research, and forensics. Today, restriction enzymes (RE) are primarily used to isolate parts of a DNA sequence (often, a gene) for cloning and replication. In cloning, restriction enzymes are used to remove a Gene Of Interest (GOI) from one organism's genome and splice it into a plasmid that can be used for mass production of a desired protein or for further manipulation/study in the laboratory. The organism containing the altered, chimeric DNA sequence, is referred to as transgenic and the transplanted GOI as a transgene. In this way, humans have been actively editing DNA since the mid 1970s and developed methods for using RFLP analysis to aid in a variety of undertakings. Recently, new exciting endonuclease enzymes (Cas9, in particular) have been discovered that progress our abilities to edit DNA in specific and multifunctional ways.

In this laboratory activity, students will learn how DNA restriction analysis can be used for forensic applications. In our case, we have samples of several suspects' DNA along with a sample discovered at a crime scene. Restriction enzymes bind to specific sequences of DNA bases and cut the DNA at a specific location within that sequence. We will be using the enzyme ApaLI which recognizes the sequence GTGCAC and cuts between the first two bases, G and the T. The DNA fragments resulting from a restriction digest can be separated by size using gel electrophoresis. In electrophoresis, fragmented DNA is pulled through a gel matrix (agarose, for DNA) by an electric current. Since DNA has a negatively charged phosphate backbone, fragments exposed to an elecrtic current will be pull towards the positive electrode. In a given amount of time, smaller fragments will travel farther than larger fragments and a size-based separation can be achieved. Due to the size and complexity of the human genome, no two sequences digested with the same restriction enzyme will produce the same set of restriction fragments. This property can be used to compare sequences and identify matching DNA samples.

Questions & Answers:

Q: How does the term "DNA Fingerprinting" apply to RFLP analysis?

A: The complexity of human DNA sequences implies that no two sequences digested with the same restriction enzyme will produce the same pattern of fragment sizes. In this way, the RFLP pattern produced is the equivalent of a fingerprint in identifying an unknown person.

Q: What is the major limitation of DNA editing using restriction enzymes?

A: Restriction enzymes only cut DNA at specific, predetermined locations. Hence, DNA editing is inexact and requires extensive pre-analysis and experimentation to execute.

Q: Why do bacteria produce enzymes designed to disrupt genetic material?

A: Bacteria (particularly so-called Gram Negative bacteria with soft outer cell walls) are extremely susceptible to uptake of environmental DNA. While this characteristic (referred to as competency) has proved extremely useful for laboratory manipulation, it is dangerous to bacterial survival. Restriction enzymes are an immune system response to possible infection by foreign DNA.

Applications to Everyday Life:

1. GMOs - Restriction enzyme digestion is used in the preparation of transgenic bacteria for producing many human necessities, such as medicines and agricultural products. Genetically modified organisms like vitamin-enriched wheat and rice have saved countless lives, and the largest GMO produced good worldwide is insulin for treatment of diabetes.
2. Medicine - Since a single nucleotide change will eliminate the binding of a restriction enzyme to a DNA sequence, RFLP analysis can be used to detect various mutations in DNA. Additionally, comparisons of DNA sequences within families can be used to identify disease related alleles.
3. Heritage analysis - Need to know who your baby's daddy is? As with the identification of a criminal suspect, RFLP analysis is used to determine genetic relationships between offspring and parents.

Photographs:

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