Sequence scanning: a method for rapid sequence acquisition from large-fragment DNA clones

A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size rang...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1996-02, Vol.93 (4), p.1694-1698
Main Authors: Nurminsky, D.I, Hartl, D.L
Format: Article
Language:English
Subjects:
Animals
Bacteria
Bacteriophages
chromosome mapping
clones
Cloning, Molecular - methods
DNA
DNA libraries
DNA, Recombinant - chemistry
Drosophila - genetics
Genetic Vectors
Genomics
Libraries
Mathematical sequences
Molecular biology
Molecular Sequence Data
phage P1
Plasmids
Polymerase Chain Reaction
sequence analysis
Sequence Analysis, DNA - methods
Sequencing
Shotguns
Templates, Genetic
Walking
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.93.4.1694