PCRsequencing

PCR sequencing refers to determining the nucleotide sequence of DNA fragments that have been amplified by polymerase chain reaction. It is used to obtain exact sequences of defined regions, verify PCR products, detect mutations, and genotype specimens. The approach includes traditional Sanger sequencing of individual amplicons as well as amplicon sequencing on next-generation sequencing platforms for multiplexed targets.

Workflow typically begins with designing primers that flank the region of interest, followed by PCR amplification of the target. The amplification product is checked for correct size and purity, then prepared for sequencing. In Sanger sequencing, the amplified DNA is subjected to cycle sequencing and read in one or both directions to produce accurate short reads that are aligned to a reference sequence. In NGS-based amplicon sequencing, multiple amplicons are pooled into a library and sequenced in parallel, generating numerous reads that are mapped to a reference or assembled de novo.

Applications include validation of mutations in clinical samples, pathogen detection and typing, targeted genotyping, and microbial community profiling through amplicons such as 16S rRNA genes. PCR sequencing is also used to verify cloned inserts, assist in forensic analysis, and support genetic variant discovery in targeted regions.

Limitations include PCR-induced errors and amplification bias, primer design challenges, risk of contamination, and read-length limitations of certain sequencing methods. Quantification in amplicon sequencing can be influenced by PCR efficiency, requiring appropriate controls. Overall, PCR sequencing remains a foundational technique for targeted, high-accuracy analysis of specific DNA regions.