Showing papers on "Sequence assembly published in 1992"
TL;DR: This work describes how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure.
Abstract: Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.
98 citations
TL;DR: Examination of vector sequences fortuitously present in the EMBL sequence database as contaminating parts of submitted sequences found a sequencing error frequency of 3.55% in this subset of release 27 of the database, and the possibility that this value may be representative for corresponding errors in the database as a whole.
Abstract: We have examined vector sequences fortuitously present in the EMBL sequence database as contaminating parts of submitted sequences, and found a sequencing error frequency of 3.55% in this subset of release 27 of the database. We discuss the possibility that this value may be representative for corresponding errors in the database as a whole.
37 citations
TL;DR: The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.
Abstract: The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.
34 citations
TL;DR: The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.
Abstract: Modified PCR amplification and direct sequencing procedures for the double-stranded genomic DNA template are described. Advantages of the approach we describe are: background artifact bands previously observed using high-molecular-weight DNA as a template were eliminated by this protocol; no gel purification or subcloning of the PCR-amplified double-stranded fragment was required prior to direct sequencing; and sequences of 300 nucleotides can be easily read even after a single loading. The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.
16 citations
Journal Article•
TL;DR: A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described, which should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.
Abstract: A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.
4 citations
07 Jan 1992
TL;DR: A new algorithm based on suffix arrays to solve the problem of reconstructing the original DNA sequence given only the fragments that may contain errors is given.
Abstract: Biological and physical limitations require that DNA be sequenced in fragments. During the process of sequencing the DNA, errors are occasionally introduced. This paper addresses the problem of reconstructing the original DNA sequence given only the fragments that may contain errors. The authors give a new algorithm based on suffix arrays to solve the problem of reconstructing the original DNA sequence. A worst-case and expected-case time analysis of the algorithm is presented. A program based on this algorithm is used to reconstruct the human beta globin region on chromosome 11 (HUMHBB in GenBank) when given a set of 300 to 500 mers drawn randomly from the HUMHBB locus. >
1 citations