Rhesus blood group

Reagents

  • PCR Materials bag
    • PCR beads (1)
  • Rh Factor 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

There are many different ways to categorise blood. The most common way is the ABO grouping, followed by the Rhesus blood factor, which is either Rh positive (Rh+) or Rh negative (Rh-).

The Rh factor is determined by a cluster of genes, including the genes D and CcEe. These D gene is responsible for the D antigen, and the CcEe gene for the Cc and Ee antigens.

A person with an Rh- blood type is unable to receive blood transfusions from an Rh+ donor, as there is a strong immune response to the D antigen.

Note: Do not rely on Bento Lab and DIY testing for medical decisions. If you need to determine your Rhesus group for medical reasons, please seek medical advice.

What are we testing?

This project explores the presence or absence of gene D from the Rh gene cluster. If the gene is present, the blood’s Rh factor is positive, as the D antigen will be on the membrane of the red blood cells.

Overview

Rh CcEe-Rh D gene variations

Several genes make up the Rh gene cluster. You will explore two genes: the CcEe gene and the D gene. CcEe is present for all genotypes and acts as a control to show that the PCR reaction was successful. The D gene on the other hand is only present for those who are Rh positive.

What are the possible results for this experiment?

There are three possible results, since the D gene can either present or absent, and two copies of the gene exist – one from each chromosome:

  • Homozygous dominant: Two copies of the D gene are present, which means the person has an Rh+ blood type.
  • Homozygous recessive:  The D gene is absent, which means the person has an Rh- blood type.
  • Heterozygous: One copies of the D gene are present, which means the person has an Rh+ blood type.

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 D gene and the CcEe gene. This will take about 120 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 both the D and CcEe gene of the Rh gene cluster. It is possible to use the same primers to amplify both the D gene and CcEe gene, as the DNA sequences are very similar (homologous).

    Because the CcEe gene is comparatively long, the duration of the annealing and extension steps have been set to 60 and 90 seconds, instead of the usual 30 seconds.

    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
      • 60 sec at 64°C
      • 90 sec at 72°C
    • 5 min 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 Rh-
    CcEe gene (1200 bp)
    This result shows a person who has no copies of the D gene, so homozygous recessive. This person will have an Rh- blood type.

    3 – Result Rh+
    CcEe gene (1200 bp), D gene (600 bp)
    This result shows a person with one or two copies of D gene, so someone who is heterozygous or homozygous dominant. This person will have an Rh+ blood type.

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