Restriction Enzymes and RFLP Analysis

Objectives of the Lab

• By the end of this lab, you should be able to:
  • Describe the normal function of a restriction enzyme in bacterial cells.
  • Locate a restriction enzyme’s recognition sequence and cut sites in a sample DNA sequence.
  • Carefully combine reagents for a successful DNA restriction digestion.

Introduction to the Experiment

• DNA of Interest: The tas2R38 gene amplified by PCR will be cut using the restriction enzyme Fnu4H1.
• A common nucleotide variant in the tas2R38 gene will produce varying numbers of DNA fragments among different students.
• Techniques Used: Agarose gel electrophoresis will help us observe the differences in number of DNA fragments, particularly focusing on Restriction Fragment Length Polymorphism (RFLP) analysis, a method for observing single nucleotide polymorphisms (SNPs).

Restriction Enzymes

• Definition: Restriction enzymes are proteins that cut double-stranded DNA at specific recognition sequences (usually 6 to 8 nucleotides long).
• Function in Bacteria:
  • They act as a defense mechanism against viral infections by cleaving foreign DNA.
  • Bacteria protect their DNA by attaching methyl groups, preventing the enzymes from cutting their own DNA, as illustrated below:
  • Example: EcoRI recognizes and cuts the sequence 5’-GAATTC-3’ but will not cut methylated self DNA.

Applications of Restriction Enzymes

• Isolating DNA Fragments: Researchers use these enzymes to isolate specific DNA sequences from larger DNA molecules.
• Constructing Recombinant DNA: Different DNA sources can be combined to create recombinant DNA, which is a method used since the 1970s.
• Protection Mechanism: The bacterial chromosome is protected due to methylation, allowing only foreign DNA to be cut.

Overview of RFLP Analysis

• Single Nucleotide Polymorphisms (SNPs): A SNP is a genetic variation where one nucleotide differs among individuals.
  • This can influence traits, disease susceptibility, and drug responses.
• Example:
  • Comparing two individuals:
  • Person 1: Contains two EcoRI sites and produces 3 fragments after digestion.
  • Person 2: Has a variant that changes an A to a G, losing one EcoRI site, resulting in only 2 fragments post digestion.

Setting Up Restriction Digestion

• Fnu4H1 Recognition Sequence:
  • Recognizes sequences where N can be any nucleotide.
  • Effective in rCutSmart™ buffer.
• Basic Components for Digestion:
  1. Sterile distilled water: Final volume 50 µL.
  2. rCutSmart™ buffer: 5 µL from a 10X concentrated solution.
  3. DNA sample: 1 µg to be cut.
  4. Restriction enzyme: 1 µL for the reaction.
• Incubation: Mix well and incubate at 37°C for 15-30 minutes.

Today’s Protocol

1. Enzyme Mix Preparation:
   • Use 2 µL of Fnu4H1 and 4 µL of Cutsmart buffer.
2. Labeling:
   • Use labeled tubes for undigested (U) and digested (D) samples.
3. Sample Preparation:
   • Add distilled water (4 µL) and PCR product (5 µL) for both U and D tubes. For D, add the enzyme mix.
4. Thermal Cycling:
   • Incubate at 37°C for 30 minutes before placing on ice.

Running the Samples on an Agarose Gel

1. Prepare DNA Ladder Solution:
   • Mix distilled water (8 µL), ladder DNA (1 µL), and loading dye (3 µL).
2. Sample Preparation:
   • Add loading dye (3 µL) to both U and D tubes.
3. Gel Loading:
   • Load the 100 bp ladder, U, and D samples into the agarose gel wells.
4. Running the Gel:
   • Ensure it’s submerged in the running buffer; observe dye migration to check gel loading.
5. Imaging Results:
   • After running for 45 minutes, turn off power, view gel under blue light, and document results by photographing.

Clean-Up Protocol

• Discard enzyme mix, PCR reactions, and clean lab equipment as instructed.
• Remember to wash hands post-experiment.