Abstract |
The present invention relates generally to genomics. More specifically, the inve tion of a nucleic acid (or a nucleotide applied to sequencing in general and, in |
to genetic analysis and, in particular, ntion relates to a method of amplifica- sequence of interest) which may be particular, to genome sequencing. |
Inventors |
Tillett; Daniel (Randwick, AU) |
Assignee |
Takara Shuzo Co., Inc. (Kyoto, JP) |
Appl. No.: 581822 |
Filed: May 20, 2002 |
PCT Filed: May 1, 2000 |
PCT NO: PCT/AU00/00391 |
PCT PUB.NO.: WO00/66768 |
PCT PUB. Date: November 9, 2000 |
Foreign Application Priority Data |
Apr 30, 1999[AU] PQ0087 |
Other References |
Shuber et al., "A Simplified Procedure f Research, 5:488-493 (1995). |
or Developing Multiplex PCRs" Genome |
Liu et al., "Thermal Asymmetric Interlac and Sequencing of Insert End Fragments f Walking", Genomics, 25:674-681 (1995). |
ed PCR: Automatable Amplification rom P1 and YAC Clones for Chromosome |
Waclaw Szybalski, "From the double-helix of large genomes", Gene, 135:279-290 (19 |
to novel approaches to the sequencing 93). |
Churcher et al., "Sequencing strategies" Centre, Wellcome Trust Genome Campus, Hi |
, Chapter 20, 517-527, The Sanger nxton, Cambridge, UK. |
Ghiso et al., "A Subset of 1200 Hexamers of cDNAs by Hexamer String Primer Walkin |
is Sufficient to Sequence over 95% g", Genomics, 17:798-799 (1993). |
Primary Examiner: Horlick; Kenneth R. |
Attorney, Agent or Firm: Leydig, Voit & |
Mayer, Ltd. |
TECHNICAL FIELD |
The present invention relates generally to genomics. More specifically, the inve tion of a nucleic acid (or a nucleotide applied to sequencing in general and, in |
to genetic analysis and, in particular, ntion relates to a method of amplifica- sequence of interest) which may be particular, to genome sequencing. |
BACKGROUND ART |
The development of methods for automated advances in bioinformatics, has revoluti in the new field of genomics--the study have been used to decipher the entire ge 7, 9, 10, 20), archea (3) and eukaryotes |
DNA sequence analysis, together with onised biology and medicine and ushered of genes and genomes. These techniques nomes of a number of bacteria (5, (6, 11). |
The traditional approach to sequencing l genome, uses a three-stage divide-and-co involves the construction of a number of DNA by randomly cutting the DNA into fra size classes, and then inserting the fra of propagation in a yeast or bacterial h |
arge genomes, including the human nquer strategy (29). The first stage clone libraries of the study organism's gments, separating these into differing gments into appropriate vectors capable ost. |
The second stage involves (a) constructi by identification of shared chromosomal bacterial artificial chromosome (YAC or for example, unique sites that can be am (PCR) (sequence-tagged sites or STSs) or (b) the construction of high-resolution subcloning YAC or BAC inserts into cosmi overlaps. |
on of a low-resolution physical map landmarks on overlapping yeast or BAC) clones. The landmarks may be, plified by polymerase chain reaction restriction-enzyme digestion sites: (sequence ready) maps by randomly d vectors and identifying their landmark |
The third and final stage involves selec cosmid clones, randomly fragmenting each into M13 phage or plasmid vectors. For e clones are sequenced and assembled to co (Kbp) cosmid insert. This random (shogun tide is sequenced about eight times. |
ting a minimally overlapping set of into small pieces, and subcloning ach cosmid approximately 800 M13 phase nstruct the sequence of the 40-kilobase ) approach is redundant as ever nucleo- |
The complexity and cost of the "divide-a development of new strategies. The Insti pioneered the direct shogun sequencing o this approach, the small fragments of ch into the M13 vector. Clones are randomly constructed by direct assembly. This who has been applied to the sequencing of a including the 1.9 Mbp genome of Haemophi of Mycoplasma genitalium (10), and the 1 chii (3). This approach eliminates the n significantly reducing the overall per b sequence. However, as with all random se problem is the requirement for a high le words, every nucleotide has to be sequen alignment, sequence contigs (clusters of The initial shotgun assembly of the H. i the generation of 11.6 Mbp of random seq coverage), and yet still contained 140 c closure (9). |
nd-conquer" approach has driven the tute for Genomic Research (TIGR) has f megabase-sized (Mbp) genomes. In romosomal DNA are cloned directly sequenced and the chromosome sequence le-genome random sequencing strategy number of bacterial and archeal genomes, lus influenzae (9), the 0.58 Mbp genome .66 Mbp genome of Methanococcus jannas- eed for any prior physical mapping, ase pair cost of producing a finished quencing approaches, the inherent vel of sequence redundancy. In other ced numerous times until, by computer aligned sequences) can be constructed. nfluenzas genome, for example, involved uence data (greater than 6-fold genome ontig gaps requiring labour intensive |
An alternative to the inherent inefficie is primer walking (25). In this procedur sequence is used to extend sequence info region. The new sequence information is the process is continued until the entir is determined. Although the primer walki large-scale sequencing projects, the nee synthesis of individual primers every 40 The use of a presynthesized library of s for the synthesis of each new primer. Un ely short primers are enormous, for exam 65,536 primers, while a complete decamer idual primers. |
ncies of random shogun sequencing e, a primer designed from a known rmation into the flanking unknown used to design the next primer, and e sequence of the region of interest ng strategy appears attractive for d for time-consuming and expensive 0 to 500 bp makes it impracticable. hort primers would avoid the requirement fortunately, libraries of even relativ- ple, a complete octamer library contains library contains over a million indiv- |
Two basic solutions have been proposed t the synthesis of large primer libraries. of the primer libraries by selecting an nonamers, or decamers (4, 12, 24, 26). T by Ligation of 6-mers (SPEL-6), involves bp or longer) by the annealing of at lea hexamers (drawn from a presynthesized li hexamers or 1024 singly degenerated hexa The annealed hexamers are joined by liba performed (15-19, 27). A number of relat have been developed, including the use o 22), or based on the ligation of self-co |
o enable primer walking and yet avoid The first involves reducing the size optimise subsets of useful octamers, he second, Sequential Primer Elongation the assembly of large primers (18 st three contiguous complementary brary of the full set of all 4096 mers) to a single stranded DNA template. tion and a standard sequencing reaction ed techniques based on this approach f hexamers but omitting ligation (21, mplementary hexamer strings (8). |
A large number of technical difficulties has prevented their wide-spread use. Sim projects have suggested that reduction o 90% affects priming flexibility and gene an octamer primer library, this results 12,000 primers, with a nonamer primer li primers. While primer libraries of this would be both expensive to construct and ators have designed smaller octamer and to 3000 primers, however, these sets are sequences with little G-C variability (1 nature is the failure of many short olig sequencing reactions, for example, in on of 121 nonamer primers worked (2). This formation of template secondary structur of the primer to the correct site (18). |
exist with both approaches which ulation studies of large sequencing f primer sets by more than 80% to ral utility (1, 26). In the case of in library sets containing 6,000 to brary requiring four times as many size are technically possible, they unwieldy to use. A number of investig- nonamer primer sets containing 1000 limited in use to protein coding 2, 14, 24). Of a more fundamental onucleotides to successfully prime e report only approximately one half common problem appears linked to the es which prevent efficient binding |
The complexity of the SPEL-6 hexamer lib for large-scale sequencing projects. In library (containing 4096 primers), this phosphorylation of the hexamer primers, or chemical denaturation of double stran in the presence of single stranded bindi before sequencing, and (5) the use of th sequencing failures are common, as the l for hexamer primer annealing also promot hairpin structures that prevent efficien the reduced library and the SPEL-6 appro belled primers, and are thus limited in chemistries. |
ation strategy has limited its utility addition to a complete hexamer primer technique requires: (1) enzymatic (2) a single-stranded DNA template ded DNA, (3) a DNA ligation reaction ng protein, (4) a deproteination step e Sequence enzyme (18). In addition, ow annealing temperature required es the formation of template secondary t primer annealing. Finally, both aches are unable to use fluorescent-la- the use of sequencing hardware and |
It is an object of the present invention one of the disadvantages of the prior ar |
to overcome or ameliorate at least t, or to provide a useful alternative. |