Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1996-07-11
1999-09-07
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, C12P 1934, C12Q 168
Patent
active
059486490
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to improvements in and relating to the amplification and identification of nucleotide base sequences, and in particular but not exclusively to improvements in and relating to the amplification and identification of nucleotide base sequences using the polymerase chain reaction (PCR).
2. Description of the Related Art
The PCR is a useful technique for amplifying genetic sequences. One application of this is the amplification of target gene sequences in biological samples from, for example, environmental, food and medical sources, etc. to allow identification of causative, pathogenic, spoilage or indicator organisms present in the sample.
Conventionally, the PCR is carried out using primers chosen or produced to amplify a particular target gene sequence within a given organism. Consequently, currently the PCR can only be used to amplify a single predetermined gene sequence at any one time and hence to confirm the presence or absence of that target sequence and the corresponding organism. Thus, when the identity of one or more organisms in a sample is to be established, it is necessary to guess or assume what sequences/organisms may be present, identify and obtain specific primers that are operable to amplify that sequence or a sequence indicative of that organism, and then conduct the experiment. Such procedures are time consuming and expensive, and somewhat unreliable.
It is an object of the present invention to obviate or mitigate one or more of the above disadvantages.
SUMMARY OF THE INVENTION
Throughout this specification the terms "unidentified sequence" and "unidentified nucleotide sequence" are used to mean that the sequence has not yet been identified in the particular sample being analysed, not necessarily one which is wholly unknown. It may be that once an unidentified sequence has been amplified and characterised it is one with which scientists are familiar.
In one aspect the invention provides a method of identifying a nucleic acid sequence in a biological sample, which method comprises using at least one pair of oligonucleotide primers to amplify the nucleic acid sequence, and characterising the amplification reaction products, characterised in that the or each pair of oligonucleotide primers is operable to enable the amplification of nucleic acid sequences from different biological sources.
The amplification method is not material to the invention. Preferably amplification is effected by the polymerase chain reaction (PCR).
The method by which amplification reaction products are characterised is also not material to the invention. Such characterisation may be effected by known techniques, e.g. sequencing, or by the use of single-strand conformation polymorphism (SSCP) analysis, or by the use of a labelled probe which binds to the amplification reaction products. One suitable system is that marketed by Perkin Elmer under the trademark TaqMan which is described in more detail below.
The method of the invention is characterised by the use of at least one pair of oligonucleotide primers operable to enable the amplification of nucleic acid sequences from different biological sources. These so-called universal primers can be selected from regions of genes which are largely or completely conserved in many different biological organisms. Pairs of universal primers may be found by routine search in various different genes. To the best of the inventors' knowledge, the use of such pairs of universal primers in order to identify the biological source of a particular nucleic acid sequence has not previously been proposed.
In another aspect the invention provides a kit for identifying a nucleic acid sequence in a biological sample by the method described which kit comprises reagents, including at least one pair of oligonucleotide primers operable to enable the amplification of nucleic acid sequences from different biological sources, for amplifying the nucleic acid sequence, and means for characterising the amplification r
REFERENCES:
patent: 4965188 (1990-10-01), Mullis et al.
patent: 5470971 (1995-11-01), Kondo et al.
Jiang et la. Chloramphenicol induces the transcription of the major cold shock gene of Escherichia coli, cspA J. Bacteriology, vol. 175 (18), pp. 5824-5828), 1993.
Francis Kevin Patrick
Stewart Gordon Sydney Anderson Birnie
Biotec Laboratories Limited
Campbell Eggerton A.
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