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DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and ...
First, DNA sequencing libraries are generated by clonal amplification by PCR in vitro. Second, the DNA is sequenced by synthesis, such that the DNA sequence is determined by the addition of nucleotides to the complementary strand rather than through chain-termination chemistry. Third, the spatially segregated, amplified DNA templates are ...
Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained ...
Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production.
Individual clones from genomic libraries can be sheared into smaller fragments, usually 500bp to 1000bp, which are more manageable for sequencing. [4] Once a clone from a genomic library is sequenced, the sequence can be used to screen the library for other clones containing inserts which overlap with the sequenced clone.
The sequencing occurs for millions of clusters at once, and each cluster has ~1,000 identical copies of a DNA insert. [12] The sequence data is analyzed by finding fragments with overlapping areas, called contigs, and lining them up. If a reference sequence is known, the contigs are then compared to it for variant identification.
In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR -based cloning and next-generation sequencing technologies based on pyrosequencing often avoid using cloning vectors.
Polony sequencing allows for a high throughput and high consensus accuracies of DNA sequencing based on a commonly available, inexpensive instrument. Also, it is a very flexible technique that enables variable application including BAC (bacterial artificial chromosome) and bacterial genome resequencing, as well as SAGE (serial analysis of gene ...
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