Method of amplification of nucleic acids

Details Method of amplification of nucleic acids Method of amplification of nucleic acids

2002-05-20

2004-05-18

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C536S024330

Reexamination Certificate

active

06737253

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to genetic analysis and, in particular, to genomics. More specifically, the invention relates to a method of amplification of a nucleic acid (or a nucleotide sequence of interest) which may be applied to sequencing in general and, in particular, to genome sequencing.
BACKGROUND ART
The development of methods for automated DNA sequence analysis, together with advances in bioinformatics, has revolutionised biology and medicine and ushered in the new field of genomics—the study of genes and genomes. These techniques have been used to decipher the entire genomes of a number of bacteria (5, 7, 9, 10, 20), archea (3) and eukaryotes (6, 11).
The traditional approach to sequencing large genomes, including the human genome, uses a three-stage divide-and-conquer strategy (29). The first stage involves the construction of a number of clone libraries of the study organism's DNA by randomly cutting the DNA into fragments, separating these into differing size classes, and then inserting the fragments into appropriate vectors capable of propagation in a yeast or bacterial host.
The second stage involves (a) construction of a low-resolution physical map by identification of shared chromosomal landmarks on overlapping yeast or bacterial artificial chromosome (YAC or BAC) clones. The landmarks may be, for example, unique sites that can be amplified by polymerase chain reaction (PCR) (sequence-tagged sites or STSs) or restriction-enzyme digestion sites: (b) the construction of high-resolution (sequence ready) maps by randomly subcloning YAC or BAC inserts into cosmid vectors and identifying their landmark overlaps.
The third and final stage involves selecting a minimally overlapping set of cosmid clones, randomly fragmenting each into small pieces, and subcloning into M13 phage or plasmid vectors. For each cosmid approximately 800 M13 phase clones are sequenced and assembled to construct the sequence of the 40-kilobase (Kbp) cosmid insert. This random (shogun) approach is redundant as ever nucleotide is sequenced about eight times.
The complexity and cost of the “divide-and-conquer” approach has driven the development of new strategies. The Institute for Genomic Research (TIGR) has pioneered the direct shogun sequencing of megabase-sized (Mbp) genomes. In this approach, the small fragments of chromosomal DNA are cloned directly into the M13 vector. Clones are randomly sequenced and the chromosome sequence constructed by direct assembly. This whole-genome random sequencing strategy has been applied to the sequencing of a number of bacterial and archeal genomes, including the 1.9 Mbp genome of
Haemophilus influenzae
(9), the 0.58 Mbp genome of
Mycoplasma genitalium
(10), and the 1.66 Mbp genome of
Methanococcus jannaschii
(3). This approach eliminates the need for any prior physical mapping, significantly reducing the overall per base pair cost of producing a finished sequence. However, as with all random sequencing approaches, the inherent problem is the requirement for a high level of sequence redundancy. In other words, every nucleotide has to be sequenced numerous times until, by computer alignment, sequence contigs (clusters of aligned sequences) can be constructed. The initial shotgun assembly of the
H. influenzas
genome, for example, involved the generation of 11.6 Mbp of random sequence data (greater than 6-fold genome coverage), and yet still contained 140 contig gaps requiring labour intensive closure (9).
An alternative to the inherent inefficiencies of random shogun sequencing is primer walking (25). In this procedure, a primer designed from a known sequence is used to extend sequence information into the flanking 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. Although the primer walking strategy appears attractive for large-scale sequencing projects, the need for time-consuming and expensive synthesis of individual primers every 400 to 500 bp makes it impracticable. The use of a presynthesized library of short primers would avoid the requirement for the synthesis of each new primer. Unfortunately, libraries of even relatively short primers are enormous, for example, a complete octamer library contains 65.536 primers, while a complete decamer library contains over a million individual primers.
Two basic solutions have been proposed to enable primer walking and yet avoid the synthesis of large primer libraries. The first involves reducing the size of the primer libraries by selecting an optimise subsets of useful octamers, nonamers, or decamers (4, 12, 24, 26). The second, Sequential Primer Elongation by Ligation of 6-mers (SPEL-6), involves the assembly of large primers (18 bp or longer) by the annealing of at least three contiguous complementary hexamers (drawn from a presynthesized library of the full set of all 4096 hexamers or 1024 singly degenerated hexamers) to a single stranded DNA template. The annealed hexamers are joined by libation and a standard sequencing reaction performed (15-19, 27). A number of related techniques based on this approach have been developed, including the use of hexamers but omitting ligation (21, 22), or based on the ligation of self-complementary hexamer strings (8).
A large number of technical difficulties exist with both approaches which has prevented their wide-spread use. Simulation studies of large sequencing projects have suggested that reduction of primer sets by more than 80% to 90% affects priming flexibility and general utility (1, 26). In the case of an octamer primer library, this results in library sets containing 6,000 to 12,000 primers, with a nonamer primer library requiring four times as many primers. While primer libraries of this size are technically possible, they would be both expensive to construct and unwieldy to use. A number of investigators have designed smaller octamer and nonamer primer sets containing 1000 to 3000 primers, however, these sets are limited in use to protein coding sequences with little G-C variability (12, 14, 24). Of a more fundamental nature is the failure of many short oligonucleotides to successfully prime sequencing reactions, for example, in one report only approximately one half of 121 nonamer primers worked (2). This common problem appears linked to the formation of template secondary structures which prevent efficient binding of the primer to the correct site (18).
The complexity of the SPEL-6 hexamer libation strategy has limited its utility for large-scale sequencing projects. In addition to a complete hexamer primer library (containing 4096 primers), this technique requires: (1) enzymatic phosphorylation of the hexamer primers, (2) a single-stranded DNA template or chemical denaturation of double stranded DNA, (3) a DNA ligation reaction in the presence of single stranded binding protein, (4) a deproteination step before sequencing, and (5) the use of the Sequence enzyme (18). In addition, sequencing failures are common, as the low annealing temperature required for hexamer primer annealing also promotes the formation of template secondary hairpin structures that prevent efficient primer annealing. Finally, both the reduced library and the SPEL-6 approaches are unable to use fluorescent-labelled primers, and are thus limited in the use of sequencing hardware and chemistries.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
SUMMARY OF THE INVENTION
The present invention relates to a method of amplification of a nucleotide sequence utilising interlaced nesting primers. The method may be used for sequencing purposes and, in particular, for genome sequencing. The method when applied to sequencing has been coined Amplification and Sequencing by Interlaced Nesting (ASIN).
According to a first aspect, the present invention provides a method of amplifying a nucleotide sequence of interest wherein the nucle

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