The process continues until the full DNA molecule is sequenced. A chemical deblocking step is then used to remove the 3’ fluorescent terminal blocking group. After each round, non-incorporated molecules are washed away. A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the chip. After each round of synthesis, a camera takes a picture of the chip. These nucleotides have a reversible fluorescent blocker so the DNA polymerase can only add one nucleotide at a time onto the DNA fragment. Next, primers and modified nucleotides are washed onto the chip. This step makes about a thousand copies of each fragment of DNA and is done by bridge amplification PCR. Once the fragments have attached, a phase called cluster generation begins. Each nanowell contains oligonucleotides that provide an anchoring point for the adapters to attach. The flow cell contains nanowells that space out fragments and help with overcrowding. The modified DNA is loaded onto a flow cell where amplification and sequencing will take place. The DNA is fragmented and adapters are added that contain segments that act as reference points during amplification, sequencing, and analysis. This works in three basic steps: amplify, sequence, and analyze. The result is a cluster of DNA forward and reverse strand clones. Each forms a new bridge (bridge amplification). A polymerase synthesizes the reverse strand. The strand bends over and attaches to a second oligo forming a bridge. The DNA attaches to the flow cell via complementary sequences. It can also be used for whole- genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein- nucleic acid interaction analysis. This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |