Overview Genomic Technologies in Clinical Diagnostics

Question 1

Describe genetic technologies currently used in the genetics laboratory

Answer 1

1) Polymerase Chain Reaction (PCR):

• PCR is a technology that amplifies DNA, making millions of copies of a specific region of interest
• It’s extremely useful in many areas, including genotyping, mutation detection, pathogen detection, etc

2) DNA Sequencing:

• Next-generation sequencing technologies allow for the rapid sequencing of large amounts of DNA, enabling whole genome sequencing, targeted sequencing, RNA sequencing, etc

3) Gene Editing Technologies:

• CRISPR-Cas9 and other gene-editing technologies allow for precise, targeted modifications to the genome

4) Microarrays:

• DNA microarrays allow for the analysis of gene expression levels for thousands of genes at once

5) Genetic Transformation:

• This technique involves introducing foreign DNA into a cell
• Genetic transformation is used in a variety of research settings, such as creating genetically modified organisms or introducing a specific gene into cells in culture

6) RNA Interference (RNAi):

• RNAi is a technique used to silence the expression of specific genes
• It involves the use of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to degrade specific messenger RNA molecules, preventing their translation into protein.

7) Flow Cytometry:

• This technique is used to sort and analyse cells based on their properties
• In genetics labs, flow cytometry is often used to sort cells based on the presence or absence of a specific marker, which can be used to separate cells that have been successfully genetically modified from those that have not

8) Quantitative PCR (qPCR):

• This is a variant of PCR that allows for the quantification of the amount of DNA present
• This is often used in gene expression studies to determine how much messenger RNA is being produced from a specific gene

9) Fluorescence in situ hybridisation (FISH):

• FISH is a technique that uses fluorescent probes to visualize specific DNA or RNA sequences in cells or tissues
• It can be used to detect specific genetic aberrations, such as deletions, duplications, or translocations

Question 2

Describe Methods to identify different types of genetic abnormalities and give examples of each technique

Answer 2

1) Karyotyping:

• This is a technique where a picture of all the chromosomes in a cell is taken
• The chromosomes are stained and arranged according to size, and any large-scale chromosomal abnormalities (such as trisomy, where there is an extra copy of a chromosome, as seen in Down syndrome) can be identified

Fluorescence in situ hybridisation (FISH):

• FISH uses fluorescently labelled DNA probes that can bind to specific sequences on chromosomes
• This method is useful for identifying chromosomal abnormalities such as translocations or deletions

3) Polymerase Chain Reaction (PCR):

• PCR can be used to amplify specific DNA sequences and can be used to identify point mutations or small deletions/insertions
• E.g. PCR is often used to test for the BRCA1 or BRCA2 mutations associated with an increased risk of breast and ovarian cancer

4) Next-Generation Sequencing (NGS):

• NGS can be used to sequence entire genomes, exomes (all coding regions), or specific panels of genes
• It can identify point mutations, small insertions or deletions, and, in some cases, larger structural variants
• NGS has been used to identify novel genetic mutations associated with various rare genetic diseases

5) Microarrays:

• DNA microarrays (also known as gene chips) can be used to assess the expression of thousands of genes simultaneously, as well as to identify copy number variations (CNVs)
• E.g. microarrays might be used to identify the CNVs often associated with autism spectrum disorder

6) Quantitative PCR (qPCR):

• This variant of PCR can be used to measure the amount of a specific DNA or RNA molecule and can be used to identify over- or under-expression of genes or amplification of genomic regions

Question 3

Describe genetics in practice in the diagnostic lab

Answer 3

1) Sample Collection:

• The process begins with the collection of a sample, which can be blood, saliva, tissue biopsy, or another suitable biological sample depending on the test being performed

2) DNA Extraction:

• The DNA is then isolated from the sample using a variety of methods
• The extracted DNA is purified and its concentration is measured

3) Genetic Testing:

• The test used depends on the suspected genetic condition and can range from single gene tests to whole genome sequencing
• Techniques such as PCR, Sanger Sequencing, Next-Generation Sequencing, microarray analysis, karyotyping, FISH, or other specialised tests can be employed

4) Data Analysis:

• This often involves comparing the patient’s DNA sequence to a reference sequence to identify any differences or mutations

5) Interpretation:

• The identified changes are then evaluated to determine if they are likely to be the cause of the patient’s symptoms
• This involves understanding the function of the gene, the effect of the identified change, and its association with the disease in question