Biochemical Techniques and Instrumentation

6.1.7. Applications

1. Selective DNA isolation: PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This helps to generate hybridization probes for Southern or Northern blotting and DNA cloning.
2. DNA sequencing: PCR is applied in DNA sequencing to determine unknown PCR-amplified sequences and isolation of a DNA sequence in recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (Escherichia coli) can be rapidly screened by PCR for correct DNA vector constructs.
3. Genetic finger printing: PCR is used for genetic fingerprinting, which is a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods. PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique can also be used to determine evolutionary relationships among organisms.
4. Forensic analysis: PCR is used in the analysis of ancient DNA of about tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.
5. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification. This technique is often applied to quantitatively determine levels of gene expression.
6. Diagnosis of diseases: PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity of at least 10,000 fold higher than other methods.
7. Identification of bacteria: PCR permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. This helps in the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
8. Virology: Viral DNA can be detected by PCR. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. The amount of virus or "viral load" in a patient can also be quantified by PCR-based DNA quantification techniques.