Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1995-04-18
1996-07-09
Jones, W. Gary
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 5, 435 6, 536 243, 536 2433, C12P 1934, C12Q 170, C12Q 168, C07H 2104
Patent
active
055344240
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a novel method for identifying a base in a target position in a DNA sequence.
In the diagnostic or forensic use of DNA analysis, full sequencing of target DNA may be unnecessary where the detection of a single base variation or mismatch is sufficient to provide the required information. Such a single base variation or mismatch may for example arise from a point mutation or, in fact, any deletion or insertion of genetic material where the detection of the first irregular base in the sequence will give the required diagnostic information. Thus, Allelic Specific PCR has been developed whereby PCR (polymerase chain reaction) is carried out on a sample using a pair of primers for the target DNA one of which is relatively short and will hybridise to one allelic locus of the DNA but not to the other allelic sequence. Failure to amplify is thus indicative that the non-hybridising allele of the DNA was present but unfortunately the conditions required to obtain reliable hybridisation to the normal DNA are difficult to achieve in practice.
It has been proposed to carry out PCR using probes hybridising to positions away from the target mutation or area of allelic variation, followed by use of a labelled probe which will not hybridise to the mutated region or area of allelic variation. However, this also commonly gives false negatives.
A method of detecting allele-specific DNA called the Ligase Chain Reaction (LCR) has recently been developed and has been reviewed by F. Barang (PCR Methods and Applications Vol.1, 5-16). Two different oligonucleotides, which hybridise adjacent to each other on complementary DNA, are required and the products of LCR need to be separated on a polyacrylamide gel before a result can be determined.
Full length sequencing, particularly solid phase sequencing, as described in WO 89/09282 gives accurate results but is more demanding and may thus not be appropriate for diagnostic screening in some instances.
The present invention is based on the concept of using a polymerase reaction on four aliquots of amplified and immobilised DNA of interest in single stranded form. Each aliquot uses the same specific extension primer and a different dideoxynucleotide but no deoxynucleotides so that only the dideoxynucleotide complementary to the base in the target position is incorporated; the target position being directly adjacent to the 3' end of the specific extension primer hybridising to the DNA. Put another way, the target position on the immobilised strand is immediately 5' of where the specific primer hybridises to the DNA. Chain extension using normal deoxynucleotides is then effected (a so-called chase reaction) using the specific primer so that the dideoxy-blocked DNA will remain unreacted while the un-blocked DNA will form double stranded DNA. Various methods may then be used to distinguish double stranded DNA from non-extended DNA, i.e. substantially single stranded DNA, and thus enable the base in the target position to be identified.
The invention thus provides a method of identification of the base in a target position in a DNA sequence wherein sample DNA is subjected to amplification; the amplified DNA is immobilised and then subjected to strand separation, the non-immobilised strand being removed and an extension primer, which hybridises to the immobilised DNA immediately adjacent to the target position, is provided; each of four aliquots of the immobilised single stranded DNA is then subjected to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide whereby only the dideoxynucleotide complementary to the base in the target position becomes incorporated; the four aliquots are then subjected to extension in the presence of all four deoxynucleotides, whereby in each aliquot the DNA which has not reacted with the dideoxynucleotide is extended to form double stranded DNA while the dideoxy-blocked DNA remains as non-extended DNA; followed by identification of the double stranded and/or non-extended DNA to indica
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Lundeberg Joakim
Uhlen Mathias
CEMU Bioteknik AB
Jones W. Gary
Rees Dianne
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