DNA Sequencing Essay Example | Topics and Well Written Essays - 4000 words

DNA Sequencing - Essay Example The Sanger methods are able to sequence best from 30-350 nucleotides and therefore genomic sequencing strategies have been developed to sequence longer DNA of interest such as the gene of interest in this plant. In the shotgun sequencing strategies, DNA often of large size is shredded into smaller fragments that can then be sequenced individually. Shredding of the DNA is done by restriction enzymes or mechanically by shearing the DNA. The sequences of these fragments are then reassembled into their original order based on overlaps. Alignment of the sequences is done by a computer program to yield the complete sequence. In Whole-genome shortgun, the DNA is obtained without prior physical map knowledge and indiscriminately sheared into fragments of 100kb which are then cloned into plasmids and transformed. The DNA inserts obtained from the plasmids are sequenced individually and consequently assembled into a long contiguous sequence. The strategy has limits due to gaps which arise duri ng assembly due to the repeats in the sequences. Another strategy is primer walking which tends to deal with whole shortgun sequencing challenges in the assembly of â€Å"gaps†. Clones carrying inserts for sequences for both sides of the gap are identified and the DNA is sequenced normally. Resultant sequence is used to design a primer downstream from the former primer position. Pairwise-end sequencing is another strategy for genome sequencing which is performed on both sides of DNA of interest as opposed to one in whole-genome shortgun.... Usually alignment of the sequences is done by a computer program to yield the complete sequence. In Whole-genome shortgun, the DNA is obtained without prior physical map knowledge and indiscriminately sheared into fragments of 100kb which are then cloned into plasmids and transformed. The DNA inserts obtained from the plasmids are sequenced individually and consequently assembled into a long contiguous sequence. The strategy has limits due to gaps which arise during assembly due to the repeats in the sequences. Another strategy is primer walking which tends to deal with whole shortgun sequencing challenges in assembly of â€Å"gaps†. . Clones carrying inserts for sequences for both sides of the gap are identified and the DNA is sequenced normally. Resultant sequence is used to design a primer downstream from the former primer position. These steps are repeated over and over until the complete sequence of the insert is elucidated. Pairwise-end sequencing (double-barrel shortgun ) is another strategy for genome sequencing which is performed on both sides of DNA of interest as opposed to one in whole-genome shortgun. It reduces â€Å"gaps† thereby minimizing assembly errors which are common in whole-gun sequencing. However it poses a huge computational challenge during assembly. DNA is shredded into 150mb fragments and inserted into BACs in hierarchical shortgun sequencing strategy. Inserts are mapped into a physical map and organized by known location â€Å"Golden Tiling Path†. Inserts are fragmented further and cloned into plasmid where they are again recovered and sequenced according to â€Å"the Golden Tiling Path†. This strategy is applied for long pieces of DNA such as whole genome or chromosome and in