ExoMeth sequencing of DNA: eliminating the need for subcloning and oligonucleotide primers.

Abstract

A method is reported for sequencing DNA based on exonuclease III digestion and strand protection by using modified nucleoside triphosphates. Up to 10 kilobases of sequence information may be obtained from each strand of a given template without subcloning. Prior knowledge of the restriction map is not important; prior knowledge of any of the sequence is not required. Nor are oligonucleotide primers needed. Double-stranded cosmids, plasmids, A phage, or linear molecules (including amplified molecules) may be used as starting material. The method creates a single-stranded template from these starting molecules, thus generating highquality sequence ladders. Most commonly used DNA polymerases may be utilized, including reverse transcriptase and T7 DNA polymerase. The approach is "ordered", so little time is wasted on redundant sequencing. nucleoside triphosphate and a modified nucleotide, such as 5-methyldeoxycytidine triphosphate (5-Me-dCTP). The DNA is digested with a frequently cutting restriction enzyme that cannot digest the DNA that has incorporated the modified nucleotide. Thus, the labeled strands are all truncated at a fixed location, producing fragments that yield sequence information by electrophoresis (Fig. 1).The method greatly extends the amount of sequence information that can be obtained from one template, thus substantially reducing the number of templates needed to sequence a given nucleic acid segment. Moreover, the sequence information can be obtained from many regions of the template in parallel. Lastly, the sequence information obtained simultaneously from many regions is "ordered" rather than "shotgun", thus allowing one to return directly to uninformative regions for further clarification. The sequencing of nucleic acids can be divided into three main tasks: generation of templates, enzymatic/chemical reactions, and identification of nucleic acid fragments that have been separated according to their length. The identification step has been automated (1, 2). The process of automating the enzymatic/chemical reaction step has begun (3, 4). However, the generation of templates currently requires numerous steps that are difficult to automate, including restriction mapping, preparing subfragments for subcloning, identifying subclones, growing bacterial cultures, and purifying nucleic acids. Such template generation steps account for >80% of the effort expended in the sequencing of nucleic acids. Unfortunately, current sequencing paradigms require the generation of a new template for each 300-500 nucleotides (nt) sequenced. Under the assumption of no overlapping sequence between templates, the sequencing of both strands of an entire mammalian genome would require at least 20 million templates 300 nt each in length. A nonordered approach, such as shotgun sequencing (5), would require the generation of 100-200 million templates.We have developed a procedure that circumvents the need for primer-binding sites and also eliminates the need to know the restriction map (Fig. 1). We call this procedu...

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

“…The OLA reaction was performed with very short reporter oligonucleotides to explore the feasibility of synthesizing a set of all possible reporter sequences that would be needed to analyze SNPs in a high throughput mode. Since ligases generally prefer 8 bases for efficient ligation (18, 19), we tested an 8‐base sequence that contained either 0 or 2 degenerate sites. If a 6+2 mer (an 8‐base sequence containing 6 defined and 2 degenerate positions) could support the OLA reaction, then it would be possible to synthesize the complete set at a reasonable cost.…”

Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium

“…The S. typhimurium uhp genes were cloned from a library of EcoRIgenerated DNA (27) for the generation of nested deletions from the ends of the insert. An average of 5.6 gel readings were obtained for each sequence character.…”

Probe mapping to facilitate transposon-based DNA sequencing.

“…Transposon-based probe mapping can also be used to increase the power of other mapping, amplification, and (27); and (iii) to supply mobile pairs of appropriate rare restriction sites for ExoMeth sequencing (8). Thus, in conjunction with improved vectors and engineered minitransposons, probe-based mapping of transposon insertions has significant potential for improving the efficiency of diverse methods for DNA analysis and characterization.…”

“…Typically, large cloned DNAs are fragmented so that relatively short pieces can be brought into juxtaposition with fixed primer binding sites for sequencing, either by subcloning or by the generation of nested deletions (5). Even larger DNA segments may be sequenced directly by oligomer walking (6), by using a library of primers (7), or by the ExoMeth method (8).…”