Map First Sequence Later or Clone by Clone Strategy, DNA Genomic, Clones, Chromosome, Genetic Maps

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Methods and Uses of Genomics and Proteomics Research Methods and Uses of Genomics and Proteomics Research - Introduction Genomics Research Methods for Whole Genome Sequencing Whole Genome Shotgun-WGS Sequencing Map First Sequence Later or Clone by Clone Strategy Construction of Whole Genome BAC Map / BAC-By-BAC Approach Building of Clone Contigs / Overlapping Series of Cloned DNA Fragments Whole Genome Sequence Data Electronic PCR (e-PCR) / Bridging the Gaps Between Mapping and Sequencing of Genomes From Genome Sequences to Function - Annotation

Home >> Biotechnology and Genomics >> Methods and Uses of Genomics and Proteomics Research >>'Maps First Sequence Later' or 'Clone by clone' Strategy

Your Ad Here	‘Map fist sequence later’ or ‘clone by clone’ strategy (also called “hierarchical shotgun sequencing”). When the whole genome sequencing work on human and other organism was initiated in late-1980s, it was decided that large segments (clones) of genomic DNA (produced by partial digestion) may first be aligned in a linear order on the chromosomes as overlapping segments, which can then be used as landmarks for sequencing data. The sequences of individual clones can thus be conveniently coalesced to obtain the DNA sequence covering an entire chromosome. Large DNA segments are cloned in BAC vectors and these BACs are used for construction of physical maps.
Such a map is described as a physical map of the genome, since the physical position of each clone on a chromosome is defined in the form of ordered BACs, unlike the genetic maps where markers are placed on a linkage map or a chromosome map on the basis of recombination frequencies rather than on the basis of physical distances. In late 1980s and early 1990s, such clone-based maps were considered necessary and useful for complete genome sequencing and were therefore prepared in several animal and plant genomes. Using these clone-based maps, whole genome sequencing was successfully completed in several eukaryotes including yeast (S. cerevisiae), a nematode (C. elegans) and a higher plant (Arabidopsis thaliana). Such clone-based maps also contributed, though partly, to the whole genome sequencing of Drosophila melanogaster, the mouse and the humans.
Once the BACs are physically mapped, the physical maps can be utilized for whole genome sequencing using the following steps : (i) BAC clones are selected from the whole genome BAC map, using suitable algorithms (software), so that minimum number of BAC clones with minimum overlapping is used to over the entire genome. This is often described as selection of minimum tilling path. In case of human genome, 10,000 to 20,000 BACs were selected to generate a working draft of human genome; (ii) BAC clones re used for subcloning, so that small inserts of a manageable size for sequencing are available in cosmid or plasmid vectors (DNA segments longer than 500-800 base pairs can not be sequenced directly in manual or automated sequencers).	Your Ad Here
These subclones are subjected to shotgun (random) sequencing without ordering them within the BAC clone, so that many subclones are sequenced to ensure sequencing of all parts of a BAC. This approach has been used to sequenced to ensure sequencing genomes of yeast and a nematode, C. elegans and also partly the genomes of fruitfly, mouse and humans. In this approach, every part of the genome is actually sequenced roughly 4-5 times to ensure that no part of the genome is left out (compare with WGS sequencing, in which 8-10 fold sequencing of the entire genome is needed). Construction of a Physical map for BAC clones to be used for 'clone by clone' strategy of whole genome sequencing

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