Octamer-primed cycle sequencing: design of an optimized primer library.

Abstract

This paper describes a novel method of primer walking using octamer oligonucleotides to prime DNA sequencing reactions. Octamer sequencing is compatible with isotopic and fluorescent sequencing chemistry, reaction conditions are optimized such that the samples can be processed in parallel, and the procedure has the potential to be automated. This strategy is faster than the traditional primer walking sequencing strategy, as the existence of a primer library allows immediate access to a primer for the next sequencing reaction, eliminating delays associated with designing and synthesizing gene-specific primers. The octamer library is comprised of optimized sequencing primers, such that octamer sequencing yields results equivalent to or better than traditional primer walking. This technology is more economical because gene-specific sequencing primers, the major cost in the reaction, are replaced by an optimized subset of frequently occurring octamers that are able to prime multiple reactions.Primer walking is a very common and effective sequencing strategy. In this procedure a primer designed from a known sequence is used to extend sequence information into a previously unknown region. The new sequence information is used to design the next primer, and the process is continued until the entire sequence of the region of interest is determined. Primer walking has advantages over other sequencing strategies: It does not require multiple cloning steps or subsequent template preparations, and it is a directed and methodical approach. The primary disadvantages of primer walking include the cost of synthesizing gene-specific primers to extend the sequence (mainly because the synthesis of each oligonucleotide produces -1000-fold excess of primer) and the time involved in designing and synthesizing these new primers. These disadvantages could be eliminated if a comprehensive library of pre-existing primers were available. A strategy for high-volume sequencing of cosmid DNAs using a primer library composed of 8-, 9-, or 10-mers was proposed (Studier 1989), whereas others have proposed synthesizing a library containing a sub- RESULTS AND DISCUSSIONThe primary consideration for designing a sequencing strategy using short oligonucleotides is defining the minimal primer length that will be recognized by the DNA polymerase and provide sufficient specificity in the sequencing reaction. Additional considerations include optimizing reaction procedures such that they are amenable to automation, effective with single-or doublestranded templates, and consistently yield high quality sequence data. Assuming random sequence composition, the size of the template directly determines the probability that a priming site will be present. The size of many doublestranded sequencing templates typically ranges from 5 to 10 kbp, or 10 to 20 kb, of target sequence. Single hexamers are insufficient to routinely prime cycle sequencing reactions (data not shown) and are likely to be present multiple times in clones of this size class [probability of e...

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“…The nucleotide sequence of all constructs was confirmed using octamer sequencing (Hardin et al, 1996). Fl y stock s and germ-line transformation.…”

It's the genes! EST access to human genome content

“…Stepwise increases in RH strains and maps should allow more precise mapping of genes to aid in disease gene identification. Technologies for sequencing entire EST clones along both strands are being devel- oped, including hexamer string(45) and single octamer primer libraries (46). A comprehensive Gene Index will require completion of 5'truncated clones and characterization of differential splicing variants, or 'spliceoforms'.…”

Octamer-primed sequencing technology: development of primer identification software

“…A complete octamer library would include 4 8 (65 536) primers, although not all octamers produce high quality sequence data. The identification of a subset of octamers, characterized by both efficient priming and frequent occurrence, and the optimization of octamer-primed reaction chemistry made possible the practical utilization of a primer library (6)(7)(8). However, after implementing the use of an octamer library, we recognized the need for a computer program to automatically identify candidate primers for subsequent sequencing reactions.…”

“…An octamer primer library was determined to be optimal, since an octamer is the shortest primer that reliably generates cycle sequencing data and provides sufficient specificity in ii sequencing reactions using double-stranded DNA templates as large as 20 kb (6). A complete octamer library would include 4 8 (65 536) primers, although not all octamers produce high quality sequence data.…”