The extension Poly(A) Test (ePAT) describes a method to determine the poly(A) tail lengths of

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...

molecule A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bio ...

s. It was developed and described by A. Jänicke et al. in 2012. The method consists of three separate steps: in the first step, the poly-adenylated RNA is hybridised to a DNA

oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...

featuring a poly-deoxythymidine sequence at its 5’ end. Klenow polymerase then catalyses elongation of the mRNA’s 3’ end, using the DNA oligonucleotide as a template. This reaction takes place at 25 °C. In the second step, reverse transcriptase synthesis extends the DNA oligonucleotides that have annealed to the mRNA’s extended 3’ end. In order to ensure that DNA oligomers hybridised to internal poly(A) sequences do not serve as primers for reverse transcription, the second step is carried out at 55 °C. A third and final step involves amplification of the newly synthesised

cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...

via

PCR PCR or pcr may refer to: Science * Phosphocreatine, a phosphorylated creatine molecule * Principal component regression, a statistical technique Medicine * Polymerase chain reaction ** COVID-19 testing, often performed using the polymerase chain r ...

. This PCR requires one gene-specific and one universal primer. Analysis of the amplicons’ lengths allows for estimation of the sequence flanked by the two primers, i.e. the poly(A) tail length of the sample mRNA. According to the authors, measurement of poly(A) tail lengths and their distribution amongst different transcripts, this method can be used to determine the cell’s translation state instead of the more tedious analysis of protein translation states.

References

{{Reflist Molecular biology techniques