Nested PCR

Nested PCR is a molecular biology technique for detecting trace amounts of DNA that is developed based on conventional PCR. PCR (polymerase chain reaction) is a technique that uses DNA polymerase to exponentially amplify DNA under specific temperature conditions. It has the advantages of being simple, fast, sensitive, specific, and diverse and is widely used in genetics, molecular biology, medicine, forensics, and other fields. However, PCR also has some limitations, such as low amplification efficiency, poor specificity, and susceptibility to contamination. To overcome these problems, various improved PCR techniques have been proposed, such as nested PCR, multiplex PCR, real-time PCR, etc. Nested PCR is one of the important improved PCR techniques, which refers to performing a second PCR reaction using the product of the first PCR reaction as a template, thereby improving the amplification efficiency and specificity. Nested PCR was first used by Saiki et al. in 1988 to detect the human immunodeficiency virus (HIV) and later became widely used in genetic disease diagnosis, pathogen detection, gene therapy, and other aspects.

The Principle of Nested PCR

The principle of nested PCR is to use two pairs of primers to perform two rounds of PCR reactions on the same target sequence, thereby improving the amplification efficiency and specificity. The two pairs of primers in nested PCR have different characteristics: outer primers bind to the outside of the target DNA, amplifying larger fragments; inner primers, or nested primers, bind to the inside of the target DNA, amplifying smaller fragments.

The first round of nested PCR uses outer primers to perform standard PCR amplification on the target DNA, usually for 15–30 cycles. The amplified product of the first round of reaction is used as the template for the second round of reaction, using inner primers to perform more precise PCR amplification, usually for 25–35 cycles. The second round of reaction can only amplify the target fragment specifically produced in the first round of reaction, and will not amplify any non-specifically produced primer dimers or other false products.

The Characteristics of Nested PCR

Nested PCR has the characteristics of high sensitivity and high specificity, but also has limitations.

Table 1. Advantages and disadvantages of nested PCR

Applications of Nested PCR

Nested PCR has a wide range of applications and significance in many fields due to its high sensitivity and high specificity for detecting trace or difficult-to-extract target DNA. Here are some examples of nested PCR applications:

Genetic disease diagnosis: Nested PCR can be used to diagnose some genetic diseases caused by gene mutations or deletions, such as β-thalassemia, hemophilia, ovarian cancer, etc. Nested PCR can improve the sensitivity and specificity of detecting these genetic variants, thereby improving the accuracy and reliability of diagnosis.

Pathogen detection: Nested PCR can be used to detect some pathogens that are present in low amounts or difficult to culture in clinical samples, such as bacteria, fungi, parasites, viruses, etc. Nested PCR can quickly identify the type and quantity of pathogens, thereby guiding clinical treatment and prevention.

Gene therapy: Nested PCR can be used to evaluate the efficacy and safety of gene therapy, such as by monitoring the efficiency and stability of gene transfer, detecting the position and frequency of gene integration, excluding the possibility of gene recombination or mutation, etc. Nested PCR can improve the quality and effect of gene therapy, thereby promoting the development and application of gene therapy.