Lecture 10- Molecular Diagnostics: History and Techniques

• Molecular Diagnostics, also known as Molecular Pathology, involves examining DNA or RNA to pinpoint specific disease indicators.
• This field applies molecular biology to conduct medical testing.
• Molecular diagnostics uses various techniques to analyze biological markers within the genome and proteome.
• Investigations are conducted on human, viral, and microbial genomes, including their encoded genes and products.

Milestones in Molecular Diagnostics

• 1953: James Watson and Francis Crick discovered the DNA Double Helix, elucidating DNA structure.
• 1983: Kary Mullis invented the Polymerase Chain Reaction (PCR), pioneering a technique to amplify targeted DNA sequences.
• 1990-2003: The Human Genome Project fully sequenced the human genome, cataloging all genes and their functions.
• 2005-present: Next-Generation Sequencing (NGS) allows the examination of genomes, transcriptomes, and epigenomes through DNA sequencing.
• 2008-present: Genome-Wide Association Studies (GWAS) are conducted to identify genetic variations linked to diseases and drug responses as part of Pharmacogenomics & Personalized Medicine.

Molecular Diagnostics: Sample Processing

• Blood Samples: Used to detect infectious agents and genetic markers in whole blood, serum, or plasma.
• Saliva and Buccal Swabs: Non-invasive samples suitable for DNA-based genetic testing, including ancestry and pharmacogenomics.
• Tissue Biopsies: Tissue samples from surgical resections/biopsies are important for molecular profiling in cancer diagnostics.
• Cell-Free DNA/RNA: DNA and RNA from plasma or bodily fluids for liquid biopsy and prenatal diagnostics.
• Other Samples: Soil, water, and food analyzed for microbial pathogens and toxins.
• Key steps in processing samples include collection, storage, quality control, and optimized extraction method.

Polymerase Chain Reaction (PCR)

• PCR amplifies a targeted DNA sequence to produce numerous copies of a given sequence.
• PCR principle is based on DNA replication, utilizing a DNA template, primers, and heat-stable DNA polymerase enzyme.
• The PCR method involves denaturation, where DNA is heated to separate strands; annealing, where specific primers bind to the DNA sequence; and extension, where DNA polymerase synthesizes new strands.

Polymerase Chain Reaction (PCR): Variations

• Conventional PCR: Used to amplify specific DNA sequences, useful for genetic testing and diagnosing infectious diseases like tuberculosis and hepatitis.
• Reverse Transcriptase PCR (RT-PCR): Detects RNA sequences and is applied in gene expression analysis for cancer, autoimmune disorders, and neurological conditions.
• Quantitative PCR (qPCR): Quantifies DNA or RNA, used for viral load monitoring in HIV, hepatitis B and C, and COVID-19.
• Advantages of PCR: High sensitivity, quick turnaround (4-8 hours), cost-effective DNA amplification, and the ability to detect less common organisms, multiplying a single nucleic acid target to detectable levels.
• Limitations of PCR: Susceptible to contamination leading to false positives, limited to amplifying short DNA sequences, and potential for primer-dimer formation causing non-specific amplification, requiring expertise in primer design.

DNA Hybridization Assays

• The assays are designed to detect, locate, and quantify DNA or RNA sequences.
• Principle: Non-amplification-based detection through annealing of single-stranded DNA or RNA probes to complementary sequences in a sample.
• Method: Involves a labeled probe molecule with a sequence complementary to a DNA or RNA target that is allowed to bind, and then detected by autoradiography, fluorescence, or chemiluminescence.
• Advantages: Longer probes increase the target specificity.
• Limitations: Conditions for hybridization need to be optimized and labelled probes increase the cost of the assay.

Southern Blotting vs Northern Blotting

• Southern blotting is used to detect DNA sequences.
• DNA fragments are transferred from agarose gel to nitrocellulose/nylon membrane and hybridized with tagged DNA probes.
• Detection of genetic mutations is performed using southern blotting.
• Northern blotting is used to detect sample RNA sequences.
• DNA fragments are transferred from agarose gel to nylon membrane and hybridized with tagged DNA/RNA probes.
• The expression level of disease markers is determined using Northern Blotting
• Molecular diagnostics can be used to detect chromosomal abnormalities using Fluorescence in situ hybridization (FISH).

Fluorescence in situ Hybridization (FISH)

• Detects and locates the presence/absence of specific DNA on chromosomes.
• Principle: Fluorescently tagged DNA probes bind to complementary target DNA, producing a fluorescent signal, patterns can reveal abnormalities.
• Advantages: Offers high specificity and spatial information within cells.
• Limitations: Labor-intensive, time-consuming with expertise in interpretation.
• Usage: Prenatal detection of aneuploidy like trisomy 21 and identifying chromosomal rearrangements, gene amplifications, and deletions in cancer biopsies.

Microarray

• It is a tool for analyzing genetic variations and other genetic interactions.
• Operates by probing for thousands of mRNA molecules simultaneously, using a labeled sample with computer analysis of binding.
• The differences in the intensity of the colours, signifies expression of genes,
• Microarray can also determine Single Nucleotide Polymorphism (SNP) Genotyping, Copy Number Variation (CNV) Analysis
• One limitation of using microarrays is that it can be limited in shelf life and expensiveness.

Next-Generation Sequencing (NGS)

• NGS is a parallel sequencing technology that is high-throughput
• The process involves generating clonally amplified DNA clusters on a solid surface, followed by sequencing-by-synthesis and sequence data analyzed.
• Steps include library preparation, where DNA/RNA is fragmented and ligated to adaptors; cluster generation on a solid surface; sequencing of DNA clusters; and data analysis.
• Identification of genetic disorders can be detected using Molecular Diagnostics such as, Carrier Screening of Cystic Fibrosis where mutations in the cystic fibrosis transmembrane are determined.

Molecular Diagnostics of Genetic Disorders

• Prenatal Screening via Cell-free DNA testing to detect fetal genetic abnormalities.
• Anmiocentesis to sample amniotic fluid between 10-14 weeks, with risk of miscarriage.
• Newborn Screening to identify early congenital metabolic, and genetic disorders.
• Detecting metabolic disorders using heel stick collection method.

Molecular Diagnostics in Oncology

• Analyzing circulating tumor DNA, molecular tools like RT-PCR, NGS, are important in better assessment and diagnosis of progression of cancer.
• Important in early detections of Cancers like breast and colorectal.
• Molecular Diagnostics can be used in treating personalized medicine, by unique molecular abnormalities.
• Personalized medicine designed to treat cancer by interrupting abnormalities
• Early Diagnosis & Detection, Management, and Treating of cancer, and detecting gene amplification or overexpression can be performed using molecular techniques.

Molecular Diagnostics in Infectious Diseases

• Pathogen Detection via Polymerase Chain Reaction (PCR) and real-time (RT-PCR).
• Used to detect and diagnose viral and bacterial infections. ex. Tuberculosis and COVID-19
• Determines the causative infectious agents.
• Viral Load Monitoring using quantitative PCR to determine treatment efficacy and progress of illness.
• Allows monitoring of viral loads (HIV, hepatitis B, and hepatitis C)
• Use detection of Antimicrobial Resistance
• Used when identifying the genes by detecting mutations, assist and determine any possible treatment.
• Through the use of COVID variants via gene sequencing to track viral outbreaks and any recent pandemic strains and vaccine production.

Molecular diagnostics uses techniques to analyze biological markers within the genome and proteome. It involves examining DNA or RNA to pinpoint specific disease indicators by applying molecular biology to conduct medical testing. This includes investigations on human, viral, and microbial genomes. Key milestones include PCR, NGS and GWAS.