Principle of PCR Based Antibody Sequencing

Antibodies are highly specific and diverse proteins widely utilized in biomedical research, diagnostics, and therapy. PCR-based antibody sequencing is esteemed for its efficiency, sensitivity, and accuracy. 

 

Antibodies consist of two heavy chains (HC) and two light chains (LC), each containing variable (V) and constant (C) regions. The variable region, responsible for antigen binding, exhibits high diversity, primarily determined by the amino acid sequence of the V region.

 

PCR is an in vitro technique used to amplify specific DNA fragments. Through the design of specific primers, PCR can selectively amplify the variable region sequences of antibody genes, providing sufficient DNA templates for subsequent sequencing.

 

Steps in PCR-Based Antibody Sequencing

1. RNA Extraction and Reverse Transcription

Total RNA is extracted from antibody-producing cells, and reverse transcriptase is used to convert mRNA into cDNA.

 

2. Primer Design

Primers are specifically designed for the variable regions of the heavy and light chains of the antibody. Primer design must consider both the conserved and diverse regions of the V region to ensure PCR specificity and efficiency.

 

3. PCR Amplification

The cDNA obtained from reverse transcription serves as a template for PCR amplification. Using the designed primers, specific antibody gene fragments are massively amplified through multiple reaction cycles. 

 

4. Product Purification and Sequencing

The amplified products are purified using electrophoresis or other methods and subsequently subjected to sequencing analysis. Common sequencing methods include Sanger sequencing and Next-Generation Sequencing (NGS). The complete sequence information of the antibody is obtained by aligning the sequencing results.

 

Advantages and Applications

1. High Throughput and Sensitivity

PCR-based antibody sequencing can process a large number of samples in a short period and detect low-abundance antibody genes, making it suitable for diversity studies and clone selection.

 

2. Precision and Specificity

The PCR technique, through the design of specific primers, can precisely amplify target genes, reducing non-specific amplification and enhancing sequencing accuracy.

 

3. Wide Applications

This technique is widely used in monoclonal antibody development, antibody library screening, antibody engineering, and immune repertoire analysis, providing robust support for antibody research.

 

MtoZ Biolabs provides integrate PCR based antibody sequencing service.