Quantitative Fluorescent PCR (QF-PCR) is a molecular biology technique used to amplify and simultaneously quantify specific DNA sequences. This method is widely used in clinical diagnostics, particularly for detecting chromosomal abnormalities, such as aneuploidies (abnormal numbers of chromosomes), and in prenatal testing. QF-PCR combines the principles of Polymerase Chain Reaction (PCR) with the use of fluorescently labelled primers, allowing for the precise quantification of DNA during the amplification process.
Key Features of QF-PCR:
Quantification:
Unlike traditional PCR, which simply amplifies DNA, QF-PCR allows for the quantification of DNA in real-time by measuring the fluorescence emitted during the amplification process. This quantification helps in determining the relative amounts of specific DNA sequences.
Fluorescent Labelling:
Primers used in QF-PCR are labeled with fluorescent dyes. As the DNA is amplified, the fluorescent signal increases, which is detected and measured by specialized equipment. The amount of fluorescence correlates with the amount of DNA present.
High Sensitivity and Specificity:
QF-PCR is highly sensitive and specific, making it ideal for detecting small amounts of DNA or subtle genetic changes.
Applications of QF-PCR:
Prenatal Testing:
QF-PCR is commonly used in prenatal testing to detect common chromosomal aneuploidies such as trisomy's 21 (Down syndrome), 18 (Edwards syndrome), and 13 (Patau syndrome). It provides rapid results, often within 24-48 hours, making it useful for early diagnosis.
Diagnosis of Genetic Disorders:
QF-PCR is used to diagnose certain genetic disorders by detecting deletions, duplications, or other chromosomal abnormalities. For example, it can be used to diagnose conditions like Turner syndrome (monosomy X) or Klinefelter syndrome (XXY).
Oncology:
In cancer diagnostics, QF-PCR can be used to detect specific genetic mutations or rearrangements associated with certain types of cancers, providing valuable information for prognosis and treatment planning.
Process of QF-PCR:
Sample Preparation:
DNA is extracted from the sample, which could be blood, amniotic fluid, or other tissues.
PCR Amplification:
The specific DNA sequences of interest are amplified using fluorescently labeled primers.
Fluorescence Detection:
As the PCR reaction progresses, the fluorescent signal is measured in real-time. The intensity of the fluorescence is directly proportional to the amount of DNA amplified.
Data Analysis:
The fluorescence data is analyzed to determine the quantity of the target DNA sequences. In the case of aneuploidy testing, the ratio of fluorescence signals from different chromosomes can indicate whether there is an abnormal number of chromosomes.
Advantages:
Speed:
QF-PCR provides rapid results, which is particularly valuable in clinical settings where timely diagnosis is critical.
Accuracy
The method is highly accurate and reliable for detecting specific genetic abnormalities.
Non-invasive:
When used for prenatal testing, QF-PCR can be performed on samples obtained through non-invasive methods, such as cell-free fetal DNA from maternal blood.
