Understanding qPCR

[Part 4]

Basic Application techniques

Relative quantification

Relative quantification describes a real-time PCR experiment in which the expression of a gene of interest in one sample is compared to expression of the same gene in another sample (i.e., treated vs. untreated). The results are expressed as fold change, increase or decrease, in expression of the treated sample in relation to the untreated sample. A normalizer gene (such as β-actin) is used as a control for experimental variability in this type of quantification. The target normalizing gene should be one that does not change in either treated or untreated samples. If the research cannot find one, then multiple normalizers can be used and then averaged.

Absolute quantification

Absolute quantification describes a real-time PCR experiment in which samples of known quantity are serially diluted and then amplified to generate a standard curve. Unknown samples are then quantified by comparison with this curve.

Allelic Discrimination

Allelic discrimination is a molecular technique used to differentiate between two alleles of a specific single nucleotide polymorphism (SNP) within a DNA sample. This process is crucial in SNP genotyping assays, allowing researchers to identify and distinguish between genetic variants.

Applications of Allelic Discrimination:

1. Pharmacogenomics: Drug Response Prediction: Identifying genetic variants that influence individual responses to drugs, aiding in personalized medicine. Adverse Drug Reactions: Detecting alleles associated with adverse drug reactions to prevent harmful side effects.
2. Genetic Research: Genotype-Phenotype Correlation: Studying the association between genetic variants and phenotypic traits to understand the genetic basis of diseases and traits. Population Genetics: Analyzing allele frequency distribution within and between populations to study genetic diversity and evolutionary patterns.
3. Clinical Diagnostics: Disease Diagnosis: Identifying genetic mutations associated with inherited diseases, such as cystic fibrosis and sickle cell anemia. Carrier Screening: Detecting carrier status for genetic disorders in prospective parents.
4. Agricultural Biotechnology: Marker-Assisted Breeding: Identifying desirable traits in plants and animals to enhance breeding programs. Genetic Modification Monitoring: Tracking the presence of genetically modified organisms (GMOs) in agricultural products.
5. Forensic Science: Human Identification: Genotyping SNPs to assist in forensic investigations and paternity testing. Sample Authentication: Verifying the authenticity of biological samples in research and clinical settings.
6. Cancer Research: Mutation Detection: Identifying somatic mutations in cancer genes to understand tumorigenesis and guide targeted therapy. Minimal Residual Disease: Monitoring genetic changes in cancer patients during and after treatment to detect minimal residual disease and relapse.

Mechanism of Allelic Discrimination in TaqMan? Technology

1. Design of Probes:

TaqMan? Probes: Two allele-specific probes are designed to target the SNP site. Each probe is labeled with a different fluorescent reporter dye at the 5' end and a non-fluorescent quencher (NFQ) at the 3' end.

VIC?-Labeled Probe: Targets and binds to Allele 1

FAM?-Labeled Probe: Targets and binds to Allele 2

2. PCR Amplification: During the polymerase chain reaction (PCR), sequence-specific primers flanking the SNP site amplify the target DNA region.

3. Hybridization and Cleavage: During the annealing phase of PCR, the TaqMan? probes hybridize to their complementary sequences at the SNP site. As DNA polymerase extends the primers, it encounters the bound probe and its 5'-nuclease activity cleaves the probe. Cleavage separates the fluorescent reporter dye from the quencher, resulting in an increase in fluorescence.

4. Fluorescence Detection: The increase in fluorescence is detected in real-time. The distinct fluorescence signals from the VIC? and FAM? dyes correspond to the presence of specific alleles. Homozygous samples will show fluorescence from only one dye, while heterozygous samples will show fluorescence from both dyes.

5. Data Analysis: The fluorescence data is plotted to create an allelic discrimination plot. This plot typically shows distinct clusters representing homozygous and heterozygous genotypes.

Homozygous for Allele 1: High VIC? signal, low FAM? signal.

Homozygous for Allele 2: High FAM? signal, low VIC? signal.

Heterozygous: High signals from both VIC? and FAM?.

Applications of qPCR technology are diverse and wide-ranging

• Gene expression analysis: Real-time PCR is widely used to study gene expression levels in various biological samples. It enables researchers to quantify the amount of specific mRNA molecules present in a sample, providing valuable insights into gene regulation and function.
• Pathogen detection: Real-time PCR is a sensitive and specific method for detecting and quantifying pathogens such as viruses, bacteria, and fungi. It is commonly used in clinical diagnostics, food safety testing, and environmental monitoring.
• Genetic variation analysis: Real-time PCR can be used to analyze genetic variations, such as single nucleotide polymorphisms (SNPs), insertions, deletions, and copy number variations. This information is useful for studying genetic diversity, disease susceptibility, and treatment response.
• Microbiome studies: Real-time PCR is employed in microbiome research to quantify the abundance of specific microbial species in complex microbial communities. It helps researchers understand the composition and dynamics of microbial populations in different environments.
• Drug discovery and development: Real-time PCR is utilized in pharmaceutical research to evaluate drug efficacy, toxicity, and pharmacogenetics. It enables researchers to assess the impact of drugs on gene expression and molecular pathways.
• Genetically modified organism (GMO) detection:?Identifying the presence of genetically modified sequences in food or crops.
• Cancer diagnostics:?Analyzing gene mutations or expression patterns associated with cancer.