How is the testing done? 

Genetic testing or confirmatory testing for Huntington’s disease is not undertaken without an appropriate referral. Detailed family and individual symptoms are carefully considered before initiating genetic testing. Upon receiving a referral, testing centers perform a crucial test called Polymerase Chain Reaction (PCR) to quantify the number of CAG repeats. 

Step 1 – Copying the Gene (PCR):

This is the starting point, a pivotal step to establish the CAG repeat number. After extracting blood, skilled technicians isolate DNA from the sample, and replicate it. After extracting DNA from your blood sample, the labs perform PCR analysis. This process replicates the target DNA sequence for further analysis. This is achieved by separating the DNA strands and creating a matching strand by introducing DNA building blocks and primer sequences to make a double helix structure that resembles the target DNA. This replication allows for a comprehensive examination of the HTT gene. Creating millions of copies of the HTT gene simplifies the quantification and comparison of the number of CAG repeats on each copy. This allows for a more accurate analysis of the genetic material. 


Figure 1: PCR cycle produces millions of copies of the target DNA at the end of each cycle.

Step 2 – Sorting Gene Pieces by Size (Gel Electrophoresis):

Now armed with millions of copies of DNA containing the HTT gene, lab technicians utilise a molecular scissor specific to the HTT gene. This scissor precisely cuts these large copies into tiny fragments. These fragments, with varying sizes due to specific cutting sites, are then sorted on a charged gel. During this step, the labs use a DNA fragment that is similar length to the HTT gene as a “ladder”, which helps them understand how long the target DNA fragments travel on the gel. While smaller molecules cover longer distances than larger ones, the distance covered is measured in reference to the ladder, who’s length is known. Analysing these molecules based on the distance travelled helps the technician comprehend the number of CAG repeats. 


Figure 1: Sorting of the DNA fragments based on the size of the fragments.  

Step 3 – Counting Gene Repeats (Analysis):

In this critical step, the fragments of the gene obtained in the previous stage are aligned by size, and the number of CAG repeats are counted. Since these fragments are all copies of a single strand of DNA, we understand that each fragment contains the same number of CAG repeats. 

This three-step process involves copying the gene, sorting gene pieces by size, and then counting gene repeats to ensure accuracy in results. Precision is crucial, and the number of gene copies under study is key to achieving precise results. Despite the meticulous nature of the testing process, it is essential to note that false positives (incorrectly indicating a person has the gene mutation associated with Huntington’s disease when they do not) and false negatives (incorrectly indicating a person does not have the gene mutation associated with Huntington’s disease when they do) are two rare mistakes that can sometimes occur. In such cases, Huntington’s specialists and neurologists reassess the individual and request the test results to be reanalysed.

With this knowledge on how genetic testing is done, click on the button below to understand how it can help you or your family members.