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 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.

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A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice

Nature ◽

10.1038/362258a0 ◽

1993 ◽

Vol 362 (6417) ◽

pp. 258-261 ◽

Cited By ~ 171

Author(s):

Andreas Schedl ◽

Lluís Montoliu ◽

Gavin Kelsey ◽

Günther Schütz

Keyword(s):

Transgenic Mice ◽

Copy Number ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Tyrosinase Gene

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Construction of a YAC Contig of 2 Megabases Around the MS1 Gene in Arabidopsis thaliana

Functional Plant Biology ◽

10.1071/pp9960453 ◽

1996 ◽

Vol 23 (4) ◽

pp. 453 ◽

Cited By ~ 3

Author(s):

RM Chapple ◽

AM Chaudhury ◽

KC Blomer ◽

LB Farrell ◽

ES Dennis

Keyword(s):

Arabidopsis Thaliana ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Rflp Markers ◽

Chromosome Walking ◽

Chromosome 5 ◽

Plant Populations ◽

Recombinant Plant ◽

Yac Contig ◽

The Relationship

The ms1 mutation of Arabidopsis thaliana causes male sterility by preventing the development of normal microspores in the developing anther. The gene is located on a region of chromosome 5 containing the RFLP markers g4111, g4560 and g21503. Using yeast artificial chromosome (YAC) libraries, we have constructed a contig of 38 YACs spanning approximately 2.1 megabases (approximately 2% of the genome) around MS1 and redefined the order of these RFLP markers. Chimeric YACs and repetitive DNA caused problems in chromosome 'walking'. A method for cloning YAC right ends by plasmid rescue was applied to arabidopsis. One YAC end contained a portion of the A. thaliana sucrose synthase gene ASUSI, hence locating this gene on chromosome 5 near MS1. Using recombinant plant populations containing ms1 and flanking markers, MS1 was localised to a 200 kb region within the YAC contig. In this contig the relationship between physical and genetic distance varied from less than 100 kb to 720 kb per centimorgan.

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Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes

Genome ◽

10.1139/g07-063 ◽

2007 ◽

Vol 50 (9) ◽

pp. 871-875 ◽

Cited By ~ 22

Author(s):

C.J. Coyne ◽

M.T. McClendon ◽

J.G. Walling ◽

G.M. Timmerman-Vaughan ◽

S. Murray ◽

...

Keyword(s):

Disease Resistance ◽

Bacterial Artificial Chromosome ◽

Pisum Sativum ◽

Plant Disease Resistance ◽

Copy Number ◽

Artificial Chromosome ◽

Pisum Sativum L ◽

A Genome ◽

Low Copy Number ◽

Bac Libraries

Pea ( Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2–0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

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Simultaneous Overexpression of Integrated Genes by Copy Number Amplification of a Mini-Yeast Artificial Chromosome

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1711.11061 ◽

2018 ◽

Vol 28 (5) ◽

pp. 821-825

Author(s):

Heo-Myung Jung ◽

Yeon-Hee Kim

Keyword(s):

Copy Number ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Copy Number Amplification

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Purification of circular YACs from yeast cells for DNA sequencing

Genome ◽

10.1139/g07-109 ◽

2008 ◽

Vol 51 (2) ◽

pp. 155-158 ◽

Cited By ~ 3

Author(s):

S.-H. Leem ◽

Y.-H. Yoon ◽

S. I. Kim ◽

V. Larionov

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Yeast Artificial Chromosome ◽

Artificial Chromosome ◽

Genome Project ◽

New Method ◽

Yeast Cells ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

The Human Genome Project

We describe a method for the purification of circular yeast artificial chromosome (YAC) DNA 120–150 kilobases (kb) in size that is of sufficient quantity and quality for restriction enzyme analysis and DNA sequencing. This method preferentially enriches for circular YAC DNA and avoids the time-consuming step of centrifugation in CsCl – ethidium bromide (EtBr) gradients. We applied this method to the purification of circular YACs carrying DNA segments that are extremely unstable in E. coli, including those that correspond to GAP2 and GAP3 on human chromosome 19. We showed that YAC DNA (GAP2 and GAP3) purified using this new method is clearly resolved in EtBr-stained gels. The sequence of YAC-GAP3 was obtained, representing the first GAP clone sequenced in YAC form. At present, it is estimated that there are more than 1000 gaps in the human genome that cannot be cloned using bacterial vectors. Thus, our new method may be very useful for completing the last stage of the human genome project.

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