Genetic sequence assay using DNA triple strand formation

A method of assaying genetic sequences comprises introducing double stranded DNA to be assayed into an aqueous buffer medium containing complexes of an anchor DNA strand anchored to a support matrix and hybridized with a reporter DNA strand having a detectable label. A portion of the anchor strand or the reporter strand consists of a selected sequence of bases capable of forming a triple helix or triple strand structure with a portion of particular double stranded DNA. The reporter strand is displaced from the complex upon formation of a triple helix or triple strand structure, and displaced reporter strands are detected to determine the presence of the particular double stranded DNA. Gene probes, anchor/reporter hybrids, selected gene sequences and kits, useful in the assay method, are provided.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method for assaying genetic sequences to 
genetic sequences useful in the method, and to the relevant components of 
a kit for the assay of such sequences. It also embodies the selection and 
use of triple helix forming regions or triple strand forming regions in a 
number of genes of clinical importance. 
The identification of genetic sequences is an important procedure in 
biological, medical and biotechnological research. Quantitative 
information about the copy number of genes in humans is particularly 
important in the early recognition of cancer and the prenatal 
identification of genetic defects. In the case of cancer cell recognition, 
such cells frequently have elevated copy numbers of regulatory genes and 
in some cases, the severity of the prognosis reflects the magnitude of the 
increase in copy number. Early detection while the copy number is still 
relatively low allows treatments that can give complete elimination of the 
cancer with minimum discomfort to the patient. The prenatal detection of 
genetic defects such as trisomy (three copies of a particular chromosome) 
or deletions or duplications of small portions can prepare parents and 
medical staff for later problems and the screening of high risk groups for 
such defects can help limit the number of births of children with low 
survival. 
2. Description of the Related Art 
A number of techniques exist for the detection of genes in human DNA but 
all of these are skilled techniques that are still principally used in the 
research laboratory environment. Southern blotting, Polymerase Chain 
Reaction and Ligase Chain Reaction have all been used to detect specific 
DNA sequences, but making the techniques quantitative is an extremely 
skilled process requiring considerable technical expertise and some 
statistical analysis. The technique described herein is part of a larger 
process that offers an easy-to-use alternative that combines sensitivity 
with reliability. 
Triple helix formation is described in a publication of Moser, H. E. and 
Dervan, P. B. (1987) Science, Vol. 238, pages 645-650. 
European Publication EP-A 0 330 185 of the present assignee describes a 
method for assaying genetic sequences in which a labelled reporter 
molecule is hybridized with a genetic probe and upon introduction of 
genetic material which more strongly hybridizes with the genetic probe, 
the reporter molecule disassociates from the genetic probe and is 
detected. 
European Publication EP-A 0 304 845 of the present assignee describes and 
claims a method of assaying gene expressions which comprises linking MRNA 
molecules to a solid substrate and contacting the solid substrate with an 
aqueous suspension of labelled microbeads to which gene probe molecules 
are linked. The gene probe molecules comprise sequences which hybridize 
with sequences comprised by and characteristic of the unknown or target 
mRNA molecules to be assayed, so that the labelled microbeads are linked 
to the target MRNA molecules on the substrate. By separating the solid 
substrate from microbeads unlinked to target mRNA molecules and detecting 
labelled, linked microbeads, the gene expressions are assayed. 
Labelled microbeads have been used in a number of analytical methods such 
as immunoassay methods. Extensive prior art concerning microbeads is 
described in U.S. Pat. Nos. 4,454,233 granted Jun. 12, 1984 and 4,436,826 
granted Mar. 13, 1984, both assigned to the present assignee. 
The method of the present invention for assaying genetic sequences is based 
on displacement of a reporter molecule hybridized to an anchored 
single-stranded DNA molecule. The displacement relies on the formation of 
a triple helical or triple strand DNA structure (triplex) between a 
portion of the sequence of the reporter and double-stranded DNA sequences 
in sample DNA. The reporter molecule in this case constitutes the "third 
strand" of the triple helix. The reporter/anchor hybrid is maintained in a 
suitable buffer in a reaction vessel. Double-stranded sample DNA is added 
to the buffer and the whole is incubated at a defined temperature. The 
free 5'-end of said reporter molecule forms a triple helical DNA segment 
with the sample DNA. The formation of the triple helix reduces the 
stability of the reporter/anchor stability and thus the reporter is 
displaced from the anchor DNA to the liquid phase where it can be assayed 
by means of the submicron particle attached to the reporter. The steps in 
this process are shown in diagrammatic form in FIG. 1. 
According to the present invention there is provided a method for assaying 
genetic sequences which comprises the formation of a weak DNA-DNA duplex 
in aqueous solution where one of the strands (the anchor strand) is 
covalently bound to a solid phase and the other (the reporter strand) has 
an assayable molecule or particle comprising assayable groups attached to 
one end. A portion of the reporter molecule is not hybridized to the 
anchor molecule and has a DNA sequence such that it can form a 
triple-helical DNA structure with a double stranded duplex DNA molecule of 
suitable sequence. This double stranded DNA molecule would be found in the 
sample DNA added to the reaction vessel. When the triple helical DNA 
structure forms, it causes the reporter molecule to be displaced from the 
reporter/anchor duplex. The amount of said reporter molecule that is 
displaced, when compared with suitable standards, will then give a measure 
of the amount of the double helical sequences present in the sample. The 
use of specific DNA sequences allows for absolute specificity in the 
displacement reaction. 
According to the present invention there is provided a method of assaying 
genetic sequences, which comprises introducing a sample containing double 
stranded DNA to be assayed into an aqueous medium containing at least one 
complex comprising an anchor DNA strand anchored to a support matrix, said 
anchor strand being hybridized with a reporter DNA strand having a 
detectable label, a portion of the anchor strand or the reporter strand 
consisting of a selected sequence of bases capable of forming a triple 
helix or triple strand structure with a portion of particular double 
stranded DNA, whereby said reporter strand is displaced from said complex 
upon formation of said triple helix or triple strand structure, and 
displaced reporter strands are detected to determine the presence of said 
particular double stranded DNA. 
According to the invention there are also provided a DNA probe, a DNA 
complex, a kit and selected gene sequences, useful for assaying genetic 
material. 
In an embodiment of the invention there also is provided a method of 
assaying genetic sequences, which comprises contacting a sample containing 
double stranded DNA to be assayed fixed to a support with an aqueous 
buffer medium containing a protein which promotes formation of a triple 
strand structure and containing at least one reporter DNA strand having a 
detectable label, a portion of the reporter strand consisting of a 
selected sequence of bases capable of forming a triple helix or triple 
strand structure with a portion of particular double stranded DNA, 
incubating the medium, washing to remove unbound reporter, and detecting 
bound reporter strands to determine the presence of said particular double 
stranded DNA. 
Prior art techniques such as Southern blotting, polymerase chain reaction 
or ligase chain reaction involve many time-consuming steps. Also since all 
the steps in the process are performed in the diagnostic laboratory, the 
staff have to be highly skilled and trained scientists. In the present 
embodiment, the assay vessel would be supplied containing the preformed 
reporter/anchor hybrid duplex. In the diagnostic laboratory, the sample 
DNA is added to the reaction vessel and incubated at a controlled 
temperature for a defined period of time. These parameters are defined in 
the production laboratory. The amount of reporter released is then 
determined using technology not part of this invention. Thus the steps 
required are simple and rapid and do not require great technical skill on 
the part of the operator. As a result, the time taken for the assay is 
significantly reduced and the cost correspondingly reduced. 
SUMMARY OF THE INVENTION 
The invention relates to a method of assaying genetic sequences, in which a 
labelled DNA molecule which can form a triple strand DNA structure with 
certain duplex DNA sequences is displaced from an anchored hybrid, and 
detection of displaced labelled DNA enables determination of the presence 
and amount of the specific duplex DNA to be assayed.