Lactose Intolerance

Reagents

  • PCR Materials bag
    • PCR beads (1)
  • Lactose Intolerance Primers (stored in the green refrigeration bag)
  • Gel electrophoresis
    • Agarose Tablet (1)
    • Safe DNA Stain
    • Loading Dye
    • 100bp DNA Ladder
    • Prepared 0.5x TBE buffer (prepared in Introduction to Gel Electrophoresis)
  • Salt. Not included.

Consumables

  • Transfer Pipette (1)
  • Pipette Tips (7)
  • Microcentrifuge Tubes (1)
  • PCR tubes (2)

Equipment

  • Shot glass or small glass. Not included.  
  • Micropipette
  • Bento Lab
  • Microwave

Abstract

What does this experiment test?

About 8% of milk is made up of lactose, a sugar molecule. Lactose is broken down by the enzyme lactase, which our bodies produce in the small intestines. But lactose tolerance after infancy is a relatively recent mutation, and lactose tolerance is not universal. For some people, lactose intolerance is related to genetics, and it occurs as the production of the lactase enzyme stops when they become an adult.

What are the genetics?

This project explores the genetic variation of the gene MCM6, which controls the expression of LCT, the gene that encodes the lactase enzyme.

Overview

MCM6 gene variations

You will explore two forms of the MCM6 gene: The T allele and the C allele.
T allele is associated with turning on the LCT gene, which means lactase will is produced.
C allele is associated with switching off the LCT gene in adulthood, which means lactase will not be made.

What are the possible results for this experiment?

There are three possible results, since the gene has two variations, and two occurrences of the gene exist – one from each chromosome:

  • Homozygous dominant: Both copies of the gene contain the T allele, which means the person can digest milk.
  • Homozygous recessive: Both copies of the gene contain the C allele, which means the person is likely to be lactose intolerant as an adult.
  • Heterozygous: One copy of the gene contain the T allele, and the other is C allele. The T allele is dominant, so the person likely to be able to digest milk as an adult.

Protocol

In this project, you will first extract human DNA from saliva. This will take about 20 minutes. After this, you will use PCR to amplify the variations of the MCM6 gene. This will take about 90 min, but most of it will be waiting time.
Finally, you will visualise the results using Gel Electrophoresis, which will take about 45 min.
At the end of each section, you can continue right away, or store your samples and continue later.


  1. DNA Extraction

    First, obtain the DNA sample. Use the DNA Extraction from saliva protocol. It will take ca 20 min, at the end of which you should have a clean DNA template sample in a PCR tube.

  2. PCR

    In this step, you will use PCR to amplify part of the MCM6 gene. The sequence of gene fragment we are copying includes the C/T mutation, that contributes to lactose intolerance. This C/T mutation is also known as a single nucleotide polymorphism (SNP) with the code rs4988235.

    The experiment uses a method called ARMS PCR. This method uses 4 primers: 2 outer primers that copy the whole fragment, and 2 internal primers, each one specific to either the C or T mutation. This allows amplification of DNA only when the target allele is present in the DNA sample.

    You will need the DNA template sample (1), a PCR tube with a mastermix bead (2) and the primer mix for this project (3).

    Set your micropipette to 20μL.

    Using a fresh pipette tip, transfer 20μL of the primer mix into the PCR tube with the mastermix bead. Then discard your tip.

    Now set your micropipette to 4μl.

    Using a fresh pipette tip, transfer 4μl of the DNA template sample from the sample tube into the PCR tube with the mastermix bead and primer mix. Then discard your tip.


    Place your PCR tube in the thermocycler block.

    Set up the thermocycler with the following PCR program:

    • 120 sec at 94°C
    • 35 cycles made of 3 steps
      • 30 sec at 94°C
      • 30 sec at 64°C
      • 30 sec at 72°C
    • 120 sec at 72°C

    If you need help operating the Bento Lab thermocycler, check the manual. You can use the PCR  preset (1), then modify (2) the program to the required settings (3) before running the program (4).

    The program will run for ca 2 hours. When it is finished, you can keep the result in the freezer, or use it right away for gel electrophoresis.

  3. Gel Electrophoresis

    Follow the Gel Electrophoresis Protocol to cast a gel and run it with your PCR result, and a 100bp ladder. This should take about 40 min.

  4. Visualising the Gel

    After the gel run has completed, you can visualise your results.

    Continue to wear gloves as you handle the gel

    Open the orange lid of the gel box, and wipe off the condensation.  

    Gently pour out the buffer, and dispose of the buffer down a drain.

    Drain disposal of TBE running buffers is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.

    Place the gel box onto the Bento Lab transilluminator surface. In order to get best visibility, you should do this in a room as dark as possible.

    Turn Bento Lab on, select the Gel Electrophoresis module, and turn on the Transilluminator light.

    Hold the orange filter lid over the gel to visualise the DNA bands. For documentation, use your mobile phone to take a clear picture of the gel. Rather than holding the lid over the gel, you can hold the lid directly in front of your camera lense.

    If the bands are faint, try to reduce the light in the room, e.g. by closing the curtains and turning off the lights.  You can also carefully take the gel out of the gel box and place it directly onto the transilluminator. Wear gloves when doing this, and be careful not to break the gel.

  5. Analysing your results

    Compare the picture of your gel to this example result, which has been run with all variations. Your sample should correspond to one of these variations.

    1 – Ladder – 100 bp DNA Ladder

    2 – Result CT – Control (440 bp), C allele (328 bp), T allele (166 bp)
    This result shows a person with a copy of both the T allele and C allele, so someone who is heterozygous. The ability to digest lactose is dominant, so the person is likely to be able to digest milk as an adult.

    3 – TT – Control (440 bp), T allele (166 bp)
    This result shows a person who has both copies of the T allele, so homozygous dominant. This person should be able to digest milk.

    4 – CC – Control (440 bp), C allele (328 bp)
    This result shows a person who has both copies of the C allele, so homozygous recessive. This person is likely to be lactose intolerant as an adult.

    After you have taken good photos of the gel for your documentation, you can dispose of the gel in your regular trash.

    Disposal of agarose gels is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.