Information AboutPyrosequencing |
| CATEGORIES ABOUT PYROSEQUENCING | |
| molecular biology | |
| biotechnology | |
| SHOPPER'S DELIGHT | |
|
Pyrosequencing, is a novel method of DNA Sequencing developed initially by Mostafa Ronaghi and co-workers in the late 1990s , then further by Biotage . The method is based on a Chemiluminescent Enzymatic Reaction , which is triggered when a molecular recognition event occurs. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it. Each time a Nucleotide , A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which results in a light signal. It is a method primarily used for Sequencing of short stretches of DNA , SNP detection and Methylation analysis. Such analyses are crucial for biological research, genetics and some medical and forensic applications. Pyrosequencing is fully automated, reliable and accurate, and large numbers of samples can be analysed in a short time. Pyrosequencing methods have been pursued to reduce costs relative to other automated sequencing methods. The technique of pyrosequencing is conducted in a five step process: Step 1: , ATP Sulfurylase , Luciferase and Apyrase , and the substrates, Adenosine 5´ Phosphosulfate (APS) and Luciferin . Step 2: The addition of one of the four Deoxynucleotide Triphosphate s ( DNTP s) initiates the second step. DNA polymerase will catalyze the incorporation of dNTP onto the template if it is complementary. It is important to note that if there is an incorporation there will be a release of Pyrophosphate (PPi) equivalent to the amount of dNTP incorporated. Step 3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and this can be analyzed in a program. Each light signal is proportional to the number of nucleotides incorporated. Step 4: To continue the sequencing the degrading of nucleotides is essential. Apyrase is a nucleotide degrading enzyme and does in this step clean the solution from all dNTP. Step 5: New nucleotides can be added and a new cycle can start. The technique has been further developed for whole genome sequencing by the company 454. To date this is the fastest sequencing method. However, a serious limitiation of the method is that read lengths are currently much shorter than those obtained by Di-deoxy Nucleotide based Chain Termination methods, which makes the process of Genome Assembly much more complicated, particularly for genomes which contain a large amount of Repetitive DNA . Pyrosequencing is therefore most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available EXTERNAL LINKS AND REFERENCES www.biotage.com www.454.com www.pyrosequencing.com http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11156611&dopt=Citation |