Many genomes as their amino acid or nucleotide sequences are available in several public databases, and the GenBank, EMBL and DDBJ are the largest repositories of such information. With the popularity of DNA sequencers, many genome projects were undertaken, and various organisms had their genome sequenced and annotated.
Vasco Azevedo, in Encyclopedia of Bioinformatics and Computational Biology, 2019 Multiple Sequence Alignment (MSA)-Based Interaction Prediction To avoid this problem, the primers used for this procedure are generally at least 24 nucleotides long.Įdson L. This situation may arise if the primer binds to more than one region within the target DNA. False priming of DNA synthesis can give erroneous and ambiguous results. Different initial primers enable sequencing of both strands one primer binds to the strand at one end of the insert, and the other primer binds to the opposite strand at the other end of the insert. To ensure that the overall sequence is correct and that there is no ambiguity regarding the identity of any nucleotide, both strands of the DNA must be sequenced. This primer-walking strategy proceeds until the entire cloned DNA is sequenced. In a similar manner, a third primer-binding site is selected, another oligonucleotide is synthesized, and the sequence of the next 500 bases is determined ( Fig. On the basis of this analysis, a second primer that is designed to hybridize to a region about 20 nucleotides upstream from the end of the acquired sequence is chemically synthesized and then used to determine the sequence of the next 500 nucleotides. In one commonly used strategy, the dideoxynucleotide sequencing reactions are carried out to determine the identity and order of the first 500 or so nucleotides of the DNA. As an example, the entire sequence of pieces of DNA longer than about 500 bp cannot typically be obtained in a single run in most sequencing systems, and therefore, if we want to sequence a whole gene, then a number of different strategies are used to obtain the complete sequence of large DNA fragments.
Although automated DNA sequencers allow much longer read than manual methods, the length of sequence that can be obtained from single run is still limited.