Genomic DNA (gDNA) is the complete set of DNA within an organism, containing all its genes and non-coding sequences. Quantitative Polymerase Chain Reaction (qPCR) is a laboratory technique for amplifying and measuring a targeted DNA molecule in real-time. gDNA qPCR combines these concepts to precisely detect and measure the quantity of specific DNA sequences within an organism’s total genomic DNA, providing a powerful tool for analyzing the genome.
Core Principles and Applications
The principle behind gDNA qPCR is the stability of genomic DNA. Unlike other molecules in the cell, gDNA is present in a fixed number of copies per cell. For instance, in diploid organisms like humans, most cells contain two copies of each chromosome, providing a stable baseline for quantification. This makes gDNA an ideal material for precisely measuring DNA sequence copy numbers.
A primary application is analyzing Copy Number Variation (CNV), which are alterations where a cell has an abnormal number of copies of a DNA section. Using gDNA qPCR, researchers can determine if a specific gene or a larger DNA segment has been deleted or duplicated. This is relevant for studying genetic disorders and cancer, where such changes in gene dosage have significant consequences.
Another use is determining transgene copy numbers in genetically modified organisms (GMOs). In biotechnology, it is necessary to know how many copies of an inserted gene are in an organism’s genome. This information influences the organism’s traits and the stability of the genetic modification. gDNA qPCR offers an accurate method to count these inserted copies.
The technique is also used for the detection and quantification of pathogens. By designing an assay that targets a unique DNA sequence of a virus or bacterium, scientists can measure the amount of pathogen DNA in a host’s sample. This measurement, known as pathogen load, helps diagnose infections, monitor disease progression, and evaluate treatment effectiveness.
Designing a gDNA qPCR Experiment
A successful gDNA qPCR experiment begins with the quality of the starting material. High-purity genomic DNA is needed, free from contaminants like proteins, RNA, or residual chemicals from the extraction process. Purity is assessed by measuring UV absorbance at different wavelengths, like the A260/280 and A260/230 ratios. The integrity of the gDNA, meaning it is not heavily fragmented, can be inspected on an agarose gel.
The design of primers and probes dictates the reaction’s specificity and efficiency. Primers must be designed to bind exclusively to the target sequence, avoiding the amplification of similar sequences like pseudogenes. The choice of detection chemistry also influences the design. SYBR Green is a dye that binds to any double-stranded DNA, while probe-based methods like TaqMan use sequence-specific probes that increase specificity.
For relative quantification, selecting a suitable reference gene is necessary. A reference gene, or housekeeping gene, should be a stable, single-copy gene present in the same number in all samples being compared. This gene serves as an internal control to normalize for variations in the amount of gDNA added to each reaction. Common reference genes include TERT and RNase P.
A well-designed experiment must include controls to ensure the validity of the results. The No Template Control (NTC) is a reaction containing all reagents except the DNA template and is used to check for contamination. Positive controls, which contain the target sequence, and negative controls, which lack it, are also included to confirm the assay is working correctly.
Data Analysis and Interpretation
The primary output from a qPCR instrument is the Quantification Cycle (Cq) value, also called the Threshold Cycle (Ct). This value represents the PCR cycle number at which the reaction’s fluorescent signal crosses a set threshold. The Cq value has an inverse relationship with the amount of starting target DNA; a lower Cq value means there was more initial template, and a higher Cq indicates less.
There are two main approaches to quantification. Absolute quantification determines the exact copy number of a target DNA sequence. This is done by running a series of standards—samples with a known DNA concentration—to generate a standard curve. By comparing the Cq value of an unknown sample to this curve, its precise copy number can be calculated.
Relative quantification is used to compare the amount of a target gene relative to a reference gene. This method, often employing the ΔΔCt calculation, determines the fold change in the copy number of the target gene in a test sample compared to a control sample. This is done after normalizing both to a stable reference gene.
For experiments using SYBR Green dye, a melt curve analysis is performed after amplification is complete. This step involves slowly heating the PCR products and measuring the change in fluorescence as the double-stranded DNA dissociates, or “melts.” The resulting melt curve verifies the reaction’s specificity. A single, specific product will have a distinct melting temperature, while non-specific products will generate additional peaks.
Key Distinctions from cDNA qPCR
While both gDNA and cDNA qPCR use the same technology, they answer different biological questions and differ in their starting material. The primary distinction is the template molecule. gDNA qPCR analyzes an organism’s entire genomic DNA, which contains both coding and non-coding regions. In contrast, cDNA qPCR starts with RNA, which is first converted into complementary DNA (cDNA) through reverse transcription.
This difference means a reverse transcription step is mandatory for cDNA qPCR but not for gDNA qPCR, and this extra step can introduce variability. The biological questions addressed by each technique are also different. gDNA qPCR is used to measure the quantity of a specific DNA sequence, answering questions about gene copy number or the presence of foreign DNA.
Conversely, cDNA qPCR measures the level of gene expression by quantifying the amount of messenger RNA (mRNA). Because RNA levels can change rapidly in response to cellular signals, cDNA qPCR provides a dynamic snapshot of gene activity. The stability of the template molecules also differs; gDNA is inherently stable, whereas RNA is more prone to degradation.