[7] Random cloning and sequencing by the M13/dideoxynucleotide chain termination method

Bankier, Alan T.; Weston, Kathleen; Barrell, Barclay G.

doi:10.1016/0076-6879(87)55009-1

Cited by 273 publications

(143 citation statements)

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“…TY medium was inoculated with 15 ,ul of a saturated culture of E. coli DKE7 grown in minimal medium and 10 ,u1 of a 10-6 dilution of the phage stock. After growth at 37°C for 6 h, single-stranded DNA was prepared (1). The entire DNA sample was then transformed into S. cerevisiae W303-1A spheroplasts selecting for Ura+.…”

Section: Methodsmentioning

confidence: 99%

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Kipling¹,

Kearsey²

1990

Mol. Cell. Biol.

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To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.Eucaryotic DNA replication initiates at multiple sites in chromosomes, an adaptation allowing the replication of a large genome in a short S phase without the need for fast fork movement. The activation of multiple replication units in the S phase is regulated both temporally and spatially (for a review, see reference 13); furthermore, the cell can ensure that its genome is replicated only once in any cell cycle by preventing initiation at any origin which has already been replicated. Replication must also be coordinated with other activities in the cell cycle, and the length of the S phase must be compatible with the overall length of the cell cycle. The requirement to complete replication before the onset of mitosis is highlighted in budding yeast cells, in which mitosis occurs almost immediately upon completion of DNA synthesis, but the mechanism allowing this coordination is not understood.In the yeast Saccharomyces cerevisiae, the replication of plasmid molecules as autonomous minichromosomes requires the presence of DNA sequences termed autonomously replicating sequence (ARS) elements (for a review, see reference 22). These elements provide an origin of replication for plasmid replication (4, 14) and appear to have a similar role in S. cerevisiae chromosomes (15,19). Comparison of the DNA sequence of ARS elements has led to the identification of a conserved 11-base-pair (bp) consensus sequence, 5'-(A/T)TTTATPuTTT(A/T)-3'. Detailed mutational analysis of four yeast ARS elements (3,8,17,23,26) has demonstrated an essential role for this core sequence; small mutations in this region can eliminate ARS function, and it is assumed that this sequence constitutes the binding site for an initiator protein. An AT-rich region situated 3' to the T-rich strand of the consensus sequence is important for origin function but shows little primary sequence similarity between ARSs. The deletion of this region can abolish or at least reduce ARS function, although small deletions or insertions in this region have little effect. It is not cl...

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Section: Methodsmentioning

confidence: 99%

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Kipling¹,

Kearsey²

1990

Mol. Cell. Biol.

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“…Nuclei were purified by sedimentation through 1. DNA sequencing and polymerase chain reactions DNA sequences were obtained using M13 subclones, generated by the sonication procedure (Bankier et al, 1987) and dideoxy chain termination reactions (Sanger et al, 1980). The data were handled and analysed by computer programs (Staden, 1986).…”

Section: Methodsmentioning

confidence: 99%

Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours.

et al. 1989

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“…In an initial shotgun phase, DNA fragments (1.4-2.2 kb) derived from a bacterial clone are subcloned into bacteriophage M13 or plasmid vectors (Bankier et al 1987) for sequencing by the chain terminator method (Sanger et al 1977) using both types of fluorescent chemistry (dye-labeled primers and dyelabeled terminators) (Prober et al 1987;Smith et al 1987;Lee et al 1992). Sequence reads are assembled into typically 2-10 contigs representing some 95% of the bacterial clone, and the assembled sequence is referred to as ''unfinished.''…”

Section: Determining the Human Genome Sequencementioning

confidence: 99%

Toward a Complete Human Genome Sequence

Centre¹,

Cente²

1998

Genome Res.

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We have begun a joint program as part of a coordinated international effort to determine a complete human genome sequence. Our strategy is to map large-insert bacterial clones and to sequence each clone by a random shotgun approach followed by directed finishing. As of September 1998, we have identified the map positions of bacterial clones covering ∼860 Mb for sequencing and completed >98 Mb (∼3.3%) of the human genome sequence. Our progress and sequencing data can be accessed via the World Wide Web (http://webace.sanger.ac.uk/HGP/ orhttp://genome.wustl.edu/gsc/).

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[7] Random cloning and sequencing by the M13/dideoxynucleotide chain termination method

Cited by 273 publications

References 10 publications

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours.

Toward a Complete Human Genome Sequence

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