Detection of nucleic acids and nucleic acid units

Details Detection of nucleic acids and nucleic acid units Detection of nucleic acids and nucleic acid units

1998-04-21

2000-10-03

Riley, Jezia

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 221, 536 243, 536 2431, 536 2432, 536 2532, 436525, 436164, 436173, 436517, 436538, 435 911, 435 912, C12Q 168, C07H 1900, C07H 2104, G01N 33553

Patent

active

061271209

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to methods for detecting the presence or absence of, and analysing the sequence of, target nucleic acids in a sample. Such methods may also be applied to target nucleic acid units such as nucleotides and nucleosides and their analogues. The invention also relates to chemical complexes for use in such methods, to a kit of reagents for use in carrying out the methods and to certain novel compounds of use in the methods.


BACKGROUND TO THE INVENTION

There are many situations in which it is necessary to detect, either qualitatively or quantitatively, the presence of nucleic acids such as DNA and RNA or their constituent nucleotides. Examples of such situations include medical diagnosis (eg, the detection of infectious agents like bacteria and viruses, the diagnosis of inherited and acquired genetic diseases and the establishment of tissue type), forensic tests in criminal investigations and paternity disputes and of course the more general attempt to sequence human and animal genes.
Techniques are already known for detecting nucleic acids and nucleic acid units. Available methods include, for instance:
a) fluorescence spectroscopy--this is technically very demanding if high sensitivities are to be achieved. In biological assays, its use tends to be complicated by autofluorescence of the analytes.
b) radiolabelling--this also requires high levels of technical skill but tends to be less sensitive than fluorescence spectroscopy. It also suffers from the obvious hazards involved in handling radioactive materials.
c) chemiluminescence--although this technique can be relatively quick to carry out, and avoids the problem of autofluorescence and the need to handle toxic substances, it is unfortunately relatively insensitive and yet is still technically demanding.
A disadvantage common to many known techniques is their need for large amounts of the target analyte, ie, their relatively low sensitivity. Often in the situations mentioned above the target is simply not available in sufficiently high concentrations. As a result, the available target material has to be amplified before its presence can be accurately detected.
Again, techniques are known for amplifying a nucleic acid. The most common is the well-known "polymerase chain reaction" ("PCR"). Alternatively, the target nucleic acid may be cloned into a biological vector such as a plasmid, a phage or the like, which is then inserted into a (typically bacterial) host cell. The host is permitted to multiply and the desired vector is "harvested" from the host cell after an appropriate period of time.
Clearly, the need for amplification makes a detection method more complex, costly and time-consuming and introduces greater potential for error and for contamination of the target material.
There is therefore a need for a nucleic acid detection method which is sensitive to relatively low target concentrations, and which can preferably be carried out directly on an unamplified sample. It is this need that the present invention addresses.
The invention provides a technique based on the principle of "surface enhanced Raman scattering" (SERS) and on a modification of that principle known as SERRS (surface enhanced resonance Raman scattering). These principles are already known and well documented, and have been used before in the detection and analysis of various target materials.
Briefly, a Raman spectrum arises because light incident on an analyte is scattered due to excitation of electrons in the analyte. "Raman" scattering occurs when an excited electron returns to an energy level other than that from which it came--this results in a change in wavelength of the scattered light and gives rise to a series of spectral lines at both higher and lower frequencies than that of the incident light. The scattered light can be detected orthogonally to the incident beam.
Normal Raman lines are relatively weak and Raman spectroscopy is therefore too insensitive, relative to other available detection methods, to be of use in chemical anal

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