Genome projects have become a large resourceful aspect of genetics and the study of biology, allowing us to work in reverse direction (gene to phenotype) and forward direction (phenotype to gene) in sequences of DNA. The use of genomic projects has lead to significant roles in identifying mutations, functions, and unidentified genes. Along with genomic projects came a variety of sequencing methods that are highly resourceful and used today.
Pyrosequencing is one of most commonly used sequencing methods that aids in detection of polymorphisms. Pyrosequencing is based on detection of synthesis reactions. The detection of a pyrophosphate groups occur once the pyrophosphate molecules are released during DNA synthesis. This only occurs as the DNA polymerase continues the addition of nucleotides to the strand being synthesized. To conduct pryrosequencing one must first use the PCR method to amplify target sequences that will later serve as templates. Pyrosequencing requires more cycles than regular PCR amplification using beads where single DNA strands are immobilized. Pyrosequencing uses about 50 cycles to ensure all the primer is used in the reaction, and requires one of the PCR primers to be biotinylated and another serving as an internal primer. One primer will be located at the end of the sequence being amplified and the internal primer will be located internal to the sequence giving the product of PCR will have their internal ends overlap (to work as primers for each other further into PCR). Once the PCR portion is completed one must ensure that there are no contaminants and should have some of the PCR samples run through a gel and have one sample as a negative control. Each bead will contain identical DNA fragments and is placed individually into a well for the sequencing reaction.
Proceeding to the next stage pryrosequencing is ready to commence. A DNA polymerase and a primer are added to each of the wells, which will prime the synthesis of a complementary DNA strand. Inside the wells there are also contain four deoxyribonucleotides (dATP, dGTP, dTTP, dCTP) being synthesized one at a time. When the nucleotides are added to the complementary base the reaction releases pyrophosphate (PPi) molecules. Two enzymes, sulfurylase and luciferase, then use the pyrophosphate molecule converting it into visible light. The emission of the visible light from the luciferase-catalyzed reaction is then detected and collected by a charge coupled device (CCD) camera imputing raw data outputs (pyrograms). The peak of the data collected represents the light signaled through the camera and is proportional to the number of nucleotides that were incorporated in the synthesis reaction. Then after the light emission is read, the enzyme apyrase degrades any unincorporated nucleotides and ATP prior to the next nucleotide to be added. The reaction is repeated in more cycles to generate reads from all the wells.
Once all the pyrograms are collected from pyrosequencing we can collectively use the data to detect things such as genetic variation, RNA allelic imbalance, DNA methylations status, and gene copy. Pyrosequencing is also essential since we can sequence large samples of DNA rapidly and accurately for any types of mutation occurrences. We can also use the pyrograms to compare sequences of regions with polymorphism events. The information obtained by this type of sequencing can lead to future advancement in medicine in understanding effects of genetic alterations that cause disease.