Shot-gun sequencing and amplification without cloning

Details Shot-gun sequencing and amplification without cloning Shot-gun sequencing and amplification without cloning

2001-05-07

2003-03-04

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06528288

ABSTRACT:

REFERENCES TO CITATIONS
A full bibliographic citation of the references cited in this application can be found in the section preceding the claims.
FIELD OF THE INVENTION
The invention relates to molecular biology methods. In particular, the invention relates to nucleic acid sequencing methods.
DESCRIPTION OF THE RELATED ART
Billions of DNA bases must be sequenced to meet the goals of the Human Genome Program. Technology must advance so that the amount of bases determined per unit of time is significantly increased, the quality of the data is highly accurate, and the cost per base is significantly decreased. Such technological advancements would enhance large sequencing projects, such as the Human Genome Project, and would benefit other types of research such as discovering and genotyping single nucleotide polymorphisms (SNPs) and gene-based drug discovery.
The current approach used in most large-scale sequencing projects is that of random sequencing of cloned shot-gun DNA fragments. In this procedure, randomly cut, overlapping nucleic acid fragments are cloned to form a library of random clones. These are sequenced. Sequence data from the library is aligned to form contiguous sequences (contigs). An 8-10 fold coverage is required to obtain sufficient overlap matching to obtain a contig. The gaps between the contigs are then filled in using primer-walking. Obtaining the gap sequences (sequences which constitute only the final few percent of the total desired sequence) requires a disproportionate effort compared to the number of nucleotides sequenced within the gaps.
Instead of using shot-gun clones, it would be very advantageous to develop a high-throughput, primer-based DNA sequencing strategy that uses primers selected from a pre-synthesized primer library. Conventional primer-based DNA sequencing requires the synthesis of a vast number of full-length primers for implementing a full-fledged primer-walking procedure. For example, conventional primer walking using 16 base long primers requires the synthesis of 4
16
primers. If a library of shorter primers could be used for this purpose, it would greatly reduce the number of primers needed for primer walking.
In 1989, Studier proposed a strategy for high-volume sequencing of cosmid DNAs using a primer library composed of 8-, 9-, or 10-mers. Others have proposed synthesizing a library containing a subset of useful octamers or nonamers (Slemieniak and Slightom, 1990; Burbelo and Iadarola, 1994). The use of ligated or non-ligated pentamer/hexamer strings has also been proposed (Kaczorowski and Szybalski, 1994; Kieleczawa, et al., 1992). A reduced library of selected nonamers has also been proposed (Siemieniak and Slightom, 1990). Several reports have demonstrated limited success with using short primer strings to prime fluorescence-based sequence reactions (Hon and Smith, 1994; Kolter, L., et al., 1994; McCombie and Kieleczawa, 1994) Bock and Slightom (1995) reported fluorescence-based cycle-sequencing with primers selected from a nonamer library. With the “PRISM”-brand T7 DNA polymerase, a commercial kit available from Perkin Elmer/Applied Biosystems, Inc. (PE/ABI) (Foster City, Calif.), Bock and Slightom reported a complete lack of success. Although reasonable results were obtained using standard oligomers (21-mers), no sequence information was generated with nonamer primers (using the same template DNA) even after testing several different template and nonamer concentrations. Bock and Slightom used the PE/ABI cycling sequencing procedure, which gave some weak results. However, even after optimizing reaction conditions for sequencing to suit the nonamers, this procedure had a success rate of only about 50%. The modified PE/ABI cycle-sequencing procedure contained some very unusual steps. For example, the use of linear and pre-denatured plasmid DNA was a must even for this low success rate. Other peculiarities associated with the procedure included the use of a low annealing temperature (20° C. for 5 min) followed by a 5-min ramp to the 60° C. extension temperature and the use of 50 cycles. According to the authors themselves, this level of success is somewhat disappointing, as they have only partially satisfied the goal of a primer library-based DNA sequencing strategy. Thus, additional improvements are needed before such a strategy can be considered practical for large-scale genome-type sequencing.
In addition to the nonamer-based cycle-sequencing method, both (1) Hardin, et al., (1996) and (2) Jones and Hardin (1998) made efforts at carrying out octamer-primed cycle-sequencing. However, as in the case of the nonamer, this is not effective for large-scale sequencing. When octamers from a 50% GC library were assayed, only five out of fourteen primers produced sequence information, resulting in an unacceptable 35.7% reaction success rate. Optimized conditions had to be used for sequencing a particular DNA template, and a set of optimized, 75% GC library had to be selected, which gave a success rate of ~73%. For this success rate, a low annealing temperature of 40° C. had to be employed, and the reaction had to be cycled for 99 rounds (instead of the usual 30 cycles). Ball, et al., (1998) have extended the use of octamer primer by tailing the primers with modified bases. The authors used, among other modified bases, 5-nitroindole in a tail, which was expected to stabilize the primers while behaving indiscriminately in base-pairing. Although this process improves the signal intensity, there were limitations. For example, only a maximum of four 5-nitroindole residues could be added. Longer tails (>6 residues) were detrimental, as they loop back on themselves, destabilizing the primer. Additionally, longer runs of 5-nitroindole residues can form secondary structures. The optimum length for the 5-nitroindole tail is 3-4 residues. This study also showed that a considerable percentage of cases required the addition of a tail to an octamer for obtaining any sequence data. A very low annealing temperature of 30° C. had to be used.
While these studies indicated that shorter oligonucleotides such as nonamer or octamer could be used for sequencing for some situations, it is clear that these approaches have severe limitations. It will be very advantageous to developing a method by which considerably longer oligonucleotides can be provided as primers, and yet the ease of availability of primers is not compromised. What is needed is a method using longer, full-length primers for cycle-sequencing when little or no sequence information of template DNA is available. What is also needed is a method using the longer, full-length primers in combination with both (1) shot-gun sequencing for obtaining the majority of the sequence and (2) primer walking for closing the gaps. This method should avoid random fragmenting and sub-cloning the DNA and avoid the need for preparing new full-length primers.
SUMMARY OF THE INVENTION
The present invention utilizes primers in which a region of the primer sequence is fixed, and, in the preferred embodiment, the remainder of the primer sequence is randomized, thereby providing an array of all the possible sequences. Accordingly, a full-length primer species will be available to bind to a particular sequence in the template DNA.
It is a principal aim of the present invention to provide a method for sequencing a long DNA molecule without fragmenting or sub-cloning the long DNA molecule.
It is a further aim of the present invention to provide a method for PCR amplifying a DNA fragment with a long-fixed sequence degenerate primer and a short-fixed sequence degenerate primer.
Yet a further aim of the present invention is to provide a method for sequencing a long DNA molecule with a primer having an arbitrary sequence handle. The handle improves the sequencing reaction.
Yet a further aim of the present invention is to provide a method for amplifying a long DNA molecule with a primer having an arbitrary sequence handle. The handle improves the amplification reaction.
The invention is directed to a method of sequencing

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