Poly A Polymerase In Next-Generation Sequencing (NGS) Preparations

Poly A polymerase (PAP) is a key enzymatic action that is responsible for adding the poly-A tail to the 3’ end of RNA molecules. This activity has a significant role in the overall stability, transport, and translation of RNA. Within the framework of NGS, however, PAP allows for the addition of the A oligo to the RNA samples prior to sequencing so that data of high quality from various RNA sources can be acquired.

The Significance of Adding Poly A Tailing

There are many advantages attached to the incorporation of poly A tail to mRNA. First of all, A tail aids in the enhancement of the stability of RNA molecules against ribonuclease activity by exonucleases. Second, the poly A tail has a role to play on translation initiation through the promotion of ribosome attachment to the mRNA. In the case of NGS preparations, poly A-tailed mRNA can easily be used for more focused gene expression analysis along with transcript variations as they can be specifically enriched.

 NGS Sample Preparation

The NGS workflows include the preparation of the RNA samples that involve the following steps: reverse transcription, CDNA construction, and amplification. It is also common to add Poly A polymerase during CDNA synthesis in order to include a poly-A tail in the final cDNA molecules. This modification is critical to library amplification and sequencing since it increases the efficiency of the steps involved in the preparation of the libraries.

Applications in RNA-Seq

PAP is also very useful in RNA sequencing (RNA-Seq) applications because it allows for more efficient enrichment for poly A-tailed mRNA. This helps in capturing a clearer transcriptome-level picture of the organism under study. Thus, such enhancement can help in determining differentially expressed genes, alternative splicing events, or even non-coding RNAs. Furthermore, including PAP in NGS preparations enable more promising data to be obtained.

Adding PAP in NGS preparations can greatly improve the quality and accuracy of the sequencing data. What’s more important, as research continues to progress, the importance of PAP for allowing scientists to investigate the transcriptome will remain major.