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 Protein biosynthesis, synthesizing a protein.
mRNA is ...
molecule
A molecule is a group of two or more atoms that are held together by Force, attractive forces known as chemical bonds; depending on context, the term may or may not include ions that satisfy this criterion. In quantum physics, organic chemi ...
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, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
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 that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...
via
PCR. 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