A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking

The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome w...

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Veröffentlicht in:Genome 2001-04, Vol.44 (2), p.305-308, Article 305
Hauptverfasser: Chibana, Hiroji, Heinecke, Elizabeth L, Beckerman, Janna L, Magee, Paul T
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container_end_page 308
container_issue 2
container_start_page 305
container_title Genome
container_volume 44
creator Chibana, Hiroji
Heinecke, Elizabeth L
Beckerman, Janna L
Magee, Paul T
description The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome) and YAC (yeast artificial chromosome).Key words: IPCR, LM-PCR, chromosome walk, genome project, contig map.
doi_str_mv 10.1139/g00-116
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subjects Base Sequence
Candida albicans
Candida albicans - genetics
chromosome walking
Chromosome Walking - methods
Chromosomes, Artificial - genetics
copy number
DNA Primers - genetics
DNA, Fungal - genetics
DNA, Fungal - isolation & purification
Escherichia coli - genetics
fosmids
Genetic Vectors
P1 ^Aartificial chromosomes
P1 artificial chromosomes
Polymerase Chain Reaction - methods
Yeast artificial chromosomes
title A system of rapid isolation of end-DNA from a small amount of fosmid DNA, with vector-based PCR for chromosome walking
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