Lecture 27- Diagnostic Testing

Explain how PCR and PCR-RFLP can be used in genetic disease diagnosis.
Interpret results of PCR and PCR-RFLP diagnostic tests.
Design a protocol for a diagnostic test, including control experiments and examples.
Describe some examples of diagnostic testing applications.

Two main approaches that are used to detect mutations:

Detecting Size Differences in PCR Products
Certain mutations change the size of the DNA fragment amplified by PCR.
- Expanded repeats: An increased number of CAG repeats in the Huntington’s disease (HD) gene increases the PCR product size.
- Deletions: A missing segment within the gene reduces the PCR product size (e.g. lysosomal storage disease).
- Method: PCR and Gel electrophoresis to visualize size differences
Detecting Changes in Restriction Sites (PCR-RFLP)
Some mutations affect recognition sites for restriction enzymes.
- Loss of a restriction site: A mutation removes a site normally cut by the enzyme (e.g. sickle cell anaemia)
- Gain of a restriction site: A mutation introduces a new site (e.g. chondrodysplasia).
- Method: PCR, Restriction enzyme digestion, Gel electrophoresis (to observe fragment patterns)

Huntington’s disease

A dominantly-inherited disorder caused by extra copies of a glutamine codon (CAG) in the Huntington’s disease gene (exon 1).
- People with 40 or more CAG repeats will develop the disease.
  - A larger number of repeats is associated with an earlier age of disease onset.
PCR can be used to distinguish these alleles based on length.
- Large band (disease).
- Small band (wild-type).

A larger number of CAG repeats is associated with an earlier age of disease onset.
- The CAG repeat mutation is unstable; repeat expansion can occur with parental(from the father) transmission.
A smaller number of CAG repeats is associated with later age of onset.

Lysosomal Storage Disorder

A metabolic disease leading to a build-up of toxic materials in cells.
Lesions occur in many cell types, including corneas (eyes), as complex sulphated sugars are not broken down in lysosomes.
- A 22 bp deletion in arylsulphatase B gene caused a frame shift and premature stop codon.

Sickle Cell Anemia

A severe hereditary form of anemia.
- A mutated hemoglobin distorts red blood cells into a crescent shape at low oxygen levels.
- Common among individuals with African ancestry.

Wild Type vs. Sickle Cell

Wild type and sickle cell forms of the beta globin gene differ by a single base.
Glutamic acid is replaced by Valine.

Procedure Order for Sickle Cell Anemia Test

Correct order of procedures:
- PCR amplify the DNA
- Cut DNA with DdeI(Restriction Endonuclease)
- Run DNA on gel

Restriction Endonucleases (Ddel)

An enzyme that cuts DNA at specific sequences

Bacterial defense system.
- Cut at a specific symmetric sequence (e.g., EcoRI cuts at GAATTC).
- Degrade invading DNA (e.g., from phages).
- Does not cut the bacteria’s own chromosome (bases are methylated).

Mutation and Restriction Enzyme Sites

The mutation deletes a DdeI restriction enzyme site.
- Wild type allele PCR product (dsDNA) is 201 bp.
- Mutant allele PCR product (dsDNA) is 376 bp.

Interpreting results

Chondrodysplasia

Malformation of the cartilage.
- Impairs normal development of many parts of the body.
- Dwarfism, barrel-shaped chest, wide-based stance, shortened neck, forelimb deformities, deformed cartilage of the trachea

Genetic Cause of Chondrodysplasia in Texel Sheep

HpyAV Recognition Site and PCR-RFLP for Chondrodysplasia

The single base pair deletion (–T) creates a new recognition site for restriction enzyme HpyAV.
PCR-RFLP can distinguish these alleles (i.e., PCR the DNA, then cut with HpyAV).