Use of the combing process for the identification of DNA...

Details Use of the combing process for the identification of DNA... Use of the combing process for the identification of DNA...

2000-05-26

2001-06-19

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100

Reexamination Certificate

active

06248537

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to methods of analyzing DNA replication, and more particularly to the use of molecular combing to facilitate the detection and identification (i.e., mapping) of origins of replication and the measurement of the dynamic and structural relationships regulating DNA replication.
Control of DNA synthesis is essential for maintaining genome stability (
1
,
2
,
3
). In higher eukaryotes, genomic instability associated with a loss of replication control and aberrant DNA synthesis is a key feature of a variety of neoplasms and genetic diseases (
4
,
5
,
28
). In yeast, an altered pattern of DNA synthesis leads to genomic abnormalities including aneuploidies and translocations (
6
,
7
). The efficient and accurate replication of the genome in eukaryotes is accomplished by the activation of multiple bidirectional origins of replication. The temporal and spatial pattern of activation, or replication program, varies according to the developmental stage (
8
). In
X. laevis
, for example, the duration of the period of DNA replication during the cell cycle depends upon replicon size, or the distribution of replication origins (
9
). However, the organization and distribution of replication origins throughout the eukaryotic genome is not well known, and consequently the regulation of the replication program in these cells is poorly understood (
10
). This is primarily due to a variety of technical and fundamental obstacles which make it difficult to study DNA replication at the genomic level (
11
). Though a number of techniques exist for studying DNA replication in both higher and lower eukaryotes, more rapid methods are needed for the quantitative analysis of the dynamics of genome duplication (
11
).
SUMMARY OF THE INVENTION
To address the question of the spatial and temporal organization of DNA replication in a genome, we have employed the molecular combing method (
12
,
13
,
14
). The method of molecular combing is described in U.S. Pat. No. 5,840,862 (Bensimon et al.), which is incorporated herein by reference. Molecular combing permits high resolution physical mapping of specific genetic loci. The technique consists of uniformly aligning and extending genomic DNA on a substrate, such as a glass coverslip. The advantage of this method is that all molecules are aligned in one direction and identically stretched. The result is an exact correlation between the measured length of the stretched molecule and its size in kilobases. Consequently, this highly reproducible and precise method provides a unique opportunity to investigate how DNA replication is coordinated with other cell-cycle events.
This invention provides methods for localizing or identifying an origin of replication in a DNA molecule. In this method, replication intermediates corresponding to sequences of DNA in any and all regions of the genome (or on episomal/extrachromosomal replicating units, e.g. plasmids, viruses, double minute chromosomes, etc.) undergoing DNA synthesis are labeled with labeled nucleotides in vivo or in vitro. In vivo, the DNA is labeled by incorporation of the labeled nucleotide during DNA synthesis at all stages of the cell cycle. In vitro, the replication intermediates are labeled using cell free extracts of any organism including extracts from HeLa cells,
Xenopus laevis
embryonic cells (egg cell extract),
Saccharomyces cerevisiae, S. pombe, Eschericia coli
, etc., to incorporate the labeled nucleotides into the replicating DNA.
The genome can be differentially labeled to identify earlier and later replicating regions by labeling the entire genome continuously with one labeled nucleotide followed by a chase using a second labeled nucleotide at later stages of replication.
Appropriately labeled DNA is then combed on a surface for in situ analysis. More specifically, after DNA replication has terminated (one effective round of genome or DNA duplication), the DNA is extracted according to established methods. The purified DNA is then placed in a buffer, such as MES buffer, at the desired pH for combing, which is generally between a pH of 5 and 8, more particularly between a pH of 5 and 6, and most preferably at a pH of about 5.5. The sample DNA is then stretched and aligned by molecular combing on a surface, such as glass. Cosmid or PCR probes are then hybridized to the combed and labeled DNA using protocols developed in the lab in order to identify a specific region of the genome. This is necessary in order to map, or localize, the replication intermediates to any given region of the genome. The hybridized DNA is then washed and prepared for detection.
Labeled DNA and hybridized probes can be detected using appropriate antibodies (specific to the differentially labeled nucleotides) conjugated to a label, such as a fluorophore, in order to permit direct visualization of the labeled and hybridized DNA in an epifluoresence microscope. The surface containing the signals are washed to remove background and non-specific detection by the antibodies. The surface is then mounted in an appropriate buffer and examined. The detected signals (replication intermediates and hybridized probes) appear as linear fluorescent signals. Images of the signals are acquired and analyzed using software developed in the lab (CartographiX,© Institut Pasteur 1995; 1997 and CI tool,©: Institut Pasteur 1999) to permit high resolution mapping.
Replication origin locations can be mapped by comparing the location of the labeled replication intermediates with respect to the hybridized probes. More specifically, measurements are made on the sizes of the flourescent replication intermediate signals and their distances from the hybridized probes in the region. Origins are identified by monitoring the bidirectional evolution of the labeled replication intermediates from a kinetics experiment involving a chase at several different successive stages of replication. Origins are located at the centerpoints of the fluorescent signals (mapping resolution is approximately 1 to 4 kb), followed by cloning of the corresponding sequence using an established physical map of the region. The cloned sequences are tested for their ability to confer autonomous replication on episomal elements (e.g., plasmids). The corresponding nucleotide sequences are then analyzed by standard bioinformatic sequence analysis programs to identify sequence motifs. In addition, a CI tool can be used to establish spatio-temporal organization of origin of replication activities during the cell cycle (CI tool©, Institut Pasteur, 1999).
In particular, the methods of the present invention for mapping an origin of replication in a DNA molecule include the steps of:
a) incubating DNA undergoing replication in the presence of labeled nucleotides, which are incorporated into the replicating DNA;
b) aligning the DNA from step a) on a substrate;
c) hybridizing the aligned DNA with a nucleotide probe; and
d) measuring the distances between the labeled nucleotides that were incorporated into the DNA during replication and the nucleotide probe to determine the location of the origin of replication with respect to the nucleotide probe.
The present invention also relates to methods of detecting an origin of replication in a DNA molecule, which include the steps of:
a) incubating DNA undergoing replication in the presence of labeled nucleotides, which are incorporated into the replicating DNA;
b) aligning the DNA from step a) on a substrate; and
c) detecting the labeled nucleotides that were incorporated into the DNA during replication, where the labeled nucleotides are located in a region that corresponds to the origin of replication.
Another aspect of the invention relates to measuring the dynamic and structural relationships that regulate DNA replication. In particular, this aspect of the invention involves a method of measuring the rate of DNA replication, which includes the steps of:
a) adding a first labeled nucleotide to a reaction mixture of replicating DNA, where the first labeled nucleotide is incorporated into the

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