Pyrosequencing of DNA #DNA #Biotech #Medtech #RubixStrategies

Introduction:

This method involves the sequencing during synthesis. This technique was developed by Pål Nyrén and Mostafa Ronaghi at the Royal Institute of Technology, Stockholm in 1996.

History:

The history of the techniques is quite interesting. Pål Nyrén wrote in his article that the idea of new sequencing technique hit to his mind during bicycling from his lab to small village Fullbourn. The idea was to follow the activity of polymerase during incorporation of nucleotide into DNA.

Principle:

The technique involves synthesis of DNA strand against a template strand. Light emitted, upon the release of pyrophosphate after nucleotide incorporation during DNA synthesis process is detected.

Methodology:

Materials required:

·         Single stranded DNA template

·         Primer

·         DNTPs

·         DNA Polymerase

·         ATP sulfurylase

·         Luciferase and apyrase with substrate Adenosine 5’phosphosulfate

·         Luciferin

Procedure:

First of all the template strand is immobilized and hybridized to a primer from which DNA polymerase will start extension. Next, other components are added into the solution. DNA polymerase will start adding the DNTPs to the 3’ end of the primer. This will result in release of pyrophosphate upon incorporation of each nucleotide. ATP sulfurylase will convert the pyrophosphate into ATP by using adenosine 5’ phosphosulfate. This ATP will be used to convert luciferin to oxyluciferin by luciferase. The oxyluciferin will produce light proportional to the amount of ATP. This light will then be analyzed through software. Apyrase enzyme will act to degrade unincorporated nucleotides and ATPs. 

Commercialization:

Venture capital founded the company Pyrosequencing AB in Uppsala, Sweden provided by Health Cap. The purpose was to commercialize reagents and machinery for sequencing short stretches of DNA by using the pyrosequencing technique. Pyrosequencing AB was also listed on the Stockholm Stock Exchange in 1999 and in 2003, was renamed to Biotag. Pyrosequencing technology was then licensed to 454 Life Sciences that developed an array-based pyrosequencing technology. This array based pyrosequencing is now considered as a platform for large-scale DNA sequencing. 

Applications:

·         Methylation analysis

Pyrosequencing determines the methylation frequencies at consecutive cpG sites. It quantifies minor changes in methylation levels.

·         Mutational analysis

It is a reliable technique for quantification and detection of sequence variation.

·         Genetic testing

Genetic testing is an important component of several researches such as detection of diseases and the underlying causes. Pyrosequencing helps to determine sequence variation insertions, deletions, SNPs, variation in gene copy number and short tandem repeats.

·         Microbial identification

This technique gives reliable data about genotype of different microbes. Similarly it allows identification of several species by use of a conserved primer.

Pros and cons:

·         High throughput

·         Sequence length can be greater than 200bp

·         Fast and real time

·         Low reagent cost for short stretches

·         Cumbersome bioinformatics

·         Error rate is high

Reginald Swift