DNA amplification of a single cell

Details DNA amplification of a single cell DNA amplification of a single cell

2001-06-18

2004-01-06

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06673541

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase application of PCT/EP99/06912 filed Sept. 17, 1999, which claims priority to EP 98 11 7745.4 filed Sept. 19, 1998 and EP 98 11 7799.1 filed Sept. 18, 1998, and which are incorporated in their entirety by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel method for the amplification of DNA, this method being particularly useful for the amplification of the DNA or the whole genome of a single cell, chromosomes or fragments thereof. The present invention further relates to the application of the method in DNA analysis for medical, forensic, diagnostic or scientific purposes, like comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), single strand conformation polymorphism analysis (SSCP), DNA sequence analysis, “loss of heterozygosity” analysis (LOH), fingerprint analysis, and/or restriction fragment length polymorphism analysis (RFLP).
2. Description of the Related Art
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
PCR (polymerase chain reaction) is an extremely powerful in vitro method for the amplification of DNA, which was initially introduced in 1985 (Saiki (1985), Science 230, 1350-1354). By repeated thermal denaturation, primer annealing and polymerase extension, PCR can amplify a single target DNA molecule to easily detectable quantities.
Although PCR was initially applied to amplify a single locus in target DNA, it is increasingly being used to amplify multiple loci simultaneously. Frequently used primers for this general amplification of. DNA are those based on repetitive sequences within the genome, which allow amplification of segments between suitable positioned repeats. Interspersed repetitive sequence PCR (IRS-PCR) has been used to create human chromosome- and region-specific libraries (Nelson (1989), Proc. Nati. Acad. Sci. USA 86, 6686-6690). In humans, the most abundant family of repeats is the Alu family, estimated to comprise 900,000 elements in the haploid genome, thus giving an average spacing of 3-4 kb (Hwu (1986), Proc. Natl. Acad. Sci. USA 83, 3875-3879). However, a major disadvantage of IRS-PCR is that repetitive sequences like Alu or L1 are not uniformly distributed throughout the genome. Alu elements, for example, are preferentially found in the light bands of human chromosomes. Therefore, such a PCR method results in a bias toward these regions while other regions are less represented and thus not amplified or an amplification can only be obtained below detectable levels. Furthermore, this technique is only applicable to those species where abundant repeat families have been identified, whereas other species such as Drosophila and less well characterized animals and plants cannot be subjected to this method.
A more general amplification than with ISR-PCR can be achieved with “degenerate oligonucleotide-primed PCR” (DOP-PCR), with the additional advantage of species independence (Telenius (1992), Genomics 13, 718-725). DOP-PCR is based on the principle of priming from short sequences specified by the 3′-end of partially degenerate oligonucleotides used, during initial low annealing temperature cycles of the PCR protocol. Since these short sequences occur frequently, amplification of target DNA proceeds at multiple loci simultaneously.
DOP-PCR can be applied for generating libraries containing a high level of single-copy sequences, provided pure and a substantial amount of DNA of interest can be obtained, e.g. flow-sorted chromosomes, microdissected chromosome bands or isolated yeast artificial chromosomes (YACs). However, DOP-PCR seems to be not capable of providing a sufficient, uniform amplification of the DNA content of a single cell (Kuukasjärvi (1997), Genes, Chromosomes & Cancer 18, 94-101).
The sensitivity of PCR allows for the analysis of a specific target DNA in a single cell (Li (1988), Nature 335, 414-417). This led to the development of preimplantation genetic disease diagnosis using single cells from early embryos (Handyside (1989), Lancet 1, 347-349) and genetic recombination analysis using a single sperm (Cui (1989), Proc. Nati. Acad. Sci. USA 86, 9389-9393) or oocyte (Cui (1992), Genomics 13, 713-717). However, in all these cases the single cell can be analyzed only once for a given target sequence and independent confirmation of the genotype of any one cell is impossible.
A method called “primer-extension preamplification” (PEP) is directed to circumvent this problem by making multiple copies of the DNA sequences present in a single cell. PEP uses a random mixture of 15-base fully degenerated oligonucleotides as primers, thereby leading to amplification of DNA sequences from randomly distributed sites. It is estimated that about 78% of the genomic sequences in a single human haploid cell can be copied no less than 30 times (Zhang (1992), Proc. Natl. Acad. Sci. USA 89, 5847-5851). However, up to now, a complete and uniform amplification of a whole genome of a single cell has not been documented with methods such as PEP.
A method called representational difference analysis (RDA) is a subtractive DNA hybridization technique that discovers the differences between paired normal and tumor genomes (Lisitsyn (1993), Science 259, 946-951). The minimal amount of DNA needed for RDA shown is 3 ng, corresponding to ≈1×10
3
cells. However, only 70% of the genomic sequences can be reproducibly amplified by RDA (Lucito (1998), Proc. Natl. Acad. Sci. USA 95, 4487-4492). Therefore, a uniform and complete amplification of the entire genome of a single cell by representational difference analysis is not possible.
BRIEF SUMMARY OF INVENTION
Therefore, considering the prior art described above, there is a demand for a method capable of substantially uniform and preferably complete amplification of genomic DNA, particularly from a single cell.
Thus, the technical problem consists in providing means and methods which comply with the needs as described above and which eliminate the above-mentioned disadvantages.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a method for the amplification of DNA, comprising the steps of
(a) providing a sample comprising DNA;
(b) digesting the DNA to be amplified with a restriction endonuclease under conditions suitable to obtain DNA fragments of similar length, wherein said restriction endonuclease-is capable of providing 5′ overhangs wherein the terminal nucleotide of the overhang is phosphorylated or 3′ overhangs wherein the terminal nucleotide of the overhang is hydroxylated on said DNA fragments,
(c) annealing at least one primer to said DNA fragments wherein
(ca) (caa) simultaneously or subsequently, oligonucleotides representing a first primer are hybridized to said 5′ overhangs on said DNA fragments of step (b) and wherein oligonucleotides representing a second primer hybridize to 3′ overhangs generated by said first primer and wherein said first and second primer are of different length;
(cab) said second primer is ligated to said 5′ overhangs; and
(cac) said first primer is removed from said DNA fragments; or
(cb) (cba) simultaneously or subsequently, oligonucleotides representing a first primer wherein the nucleotide at the 5′ terminus is phosphorylated are hybridized to said 5′ overhangs on said DNA fragments of step (b) and wherein oligonucleotides representing a second primer hybridize with said first primer; and
(cbb) said first and second primer are ligated to said DNA fragments; or
(cc) (cca) oligonucleotides representing said primer are hybridized to said 3′ overhangs so that 5′

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