DNA Sequencing Frederick Sanger is among the rare breed of individuals, who won the Nobel Prize twice - once in for identifying amino acid sequences in proteins and the second time in for DNA sequencing. The following summarizes his contribution to the method carrying his name see Figure 08a:
Trivedi June 2, There are essentially two ways to Shotgun sequencing a genome. The Shotgun sequencing approach, also referred to as the map-based method, evolved from procedures developed by a number of researchers during the late s and 90s and that continues to develop and change.
The shotgun method was developed by GNN president J.
A primer on the two approaches to sequencing follows. View larger The human body has about trillion cells. Each chromosome is one long string of DNA that is tightly coiled in a compact bundle.
Finding the order, or sequence, of these four letters is the goal of genomics. The entire human genome is made up of about 3. To read the DNA, the chromosomes are cut into tiny pieces, each of which is read individually.
When all the segments have been read they are assembled in the correct order. Two approaches have been used to sequence the genome. They differ in the methods they use to cut up the DNA, assemble it in the correct order, and whether they map the chromosomes before decoding the sequence.
A second, newer method is called whole genome shotgun sequencing. Constructing a map requires cutting the chromosomes into large pieces and figuring out the order of these big chunks of DNA before taking a closer look and sequencing all the fragments.
Whole Genome Shotgun Sequencing The shotgun sequencing method goes straight to the job of decoding, bypassing the need for a physical map.
Therefore, it is much faster. Several copies of the genome are randomly cut into pieces that are aboutbase pairs bp long. Multiple copies of the genome are randomly shredded into pieces that are 2, base pairs bp long by squeezing the DNA through a pressurized syringe.
This is done a second time to generate pieces that are 10, bp long. Each of thesebp fragments is inserted into a BAC-a bacterial artificial chromosome.
Each 2, and 10, bp fragment is inserted into a plasmid, which is a piece of DNA that can replicate in bacteria. The two collections of plasmids containing 2, and 10, bp chunks of human DNA are known as plasmid libraries. These pieces are fingerprinted to give each piece a unique identification tag that determines the order of the fragments.
Fingerprinting involves cutting each BAC fragment with a single enzyme and finding common sequence landmarks in overlapping fragments that determine the location of each BAC along the chromosome. Then overlapping BACs with markers everybp form a map of each chromosome.
This step not needed in shotgun sequencing Each BAC is then broken randomly into 1, bp pieces and placed in another artificial piece of DNA called M This collection is known as an M13 library. This step not needed in shotgun sequencing All the M13 libraries are sequenced.
Both the 2, and the 10, bp plasmid libraries are sequenced. Sequencing both ends of each insert is critical for the assembling the entire chromosome.
These sequences are fed into a computer program called PHRAP that looks for common sequences that join two fragments together. Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each chromosome.
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In genetics, shotgun sequencing is a method used for sequencing long DNA strands.
It is named by analogy with the rapidly expanding, quasi-random firing pattern of a shotgun.. The chain termination method of DNA sequencing ("Sanger sequencing") can only be used for short DNA strands of to base kaja-net.com to this size limit, longer sequences are subdivided into smaller fragments that.
Sequencing Glossary & taxonomy Evolving Terminologies for Emerging Technologies Comments? Suggestions Revisions? Mary Chitty MSLS [email protected] Last revised September 06, The facility was designed specifically for flexibility and high throughput with a particular emphasis on efficiency and rapid scale-up.
Research is carried out on the latest instrumentation, with data collected and analyzed on one of the most innovative and flexible bioinformatics computing facilities in the world.
The human genome sequence has profoundly altered our understanding of biology, human diversity, and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past 10 years.
DNA is a long and thin macromolecule with length up to 2 meters but only cm in width. It is invisible to the naked eyes. However, the atomic force microscope (AFM) has been used since the mid s to produce topographic maps of nanostructures including DNA.