Isolation of nucleic acid

Details Isolation of nucleic acid Isolation of nucleic acid

1996-10-28

2000-07-18

Riley, Jezia

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 221, 536 2541, 536 2542, 435 6, 435 912, 435 911, 436533, 436 63, 436174, 436403, 436526, 436532, C07H 2100, C12Q 168, G01N 33553, G01N 33546

Patent

active

060909358

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to the isolation of nucleic acid, especially DNA, and in particular to a method for preparing nucleic acid samples for subsequent use in amplification procedures.
Techniques for the amplification of nucleic acids have recently revolutionised molecular biology and are now established as an indispensable tool in many procedures, for example in the detection and diagnosis of genetic and infectious diseases, in forensic medicine, in the identification of new genes or allelic variations or mutations, and in aiding routine genetic manipulations e.g. sequencing.
However, whilst new and improved amplification procedures are continually being developed, there are in certain cases particular drawbacks which limit the utility of the technique. Thus for example, DNA-based diagnostic techniques relying on DNA amplification, e.g. by the polymerise chain reaction (PCR), to detect the presence of microbial genes have proved useful in detecting bacterial and viral infectious agents and pathogenes and are rapidly acquiring importance. Nevertheless, in view of certain problems the techniques are not suitable for all diagnostic uses. One major problem is that amplification techniques such as PCR cannot be used directly on clinical samples, notably faeces or blood, which contain substances which inhibit the amplification enzymes, e.g. polymerases. The presence of red blood cells or haemoglobin presents a particular problem, and these generally need to be removed. A similar problem applies in the case of detecting microbial contamination in food samples, which also often contain inhibitory substances.
For successful amplification, the sample either has to be diluted very many times or the DNA has to be isolated and purified from the sample. In the former case, dilution to a degree sufficient to permit amplification frequently entails an unacceptable loss of sensitivity. In the latter case, nucleic acid purification techniques, involving for example extraction with phenols, chloroform and alcohols are often tedious, complicated and time consuming and may lead to loss of sample DNA, which can be a problem if the sample is small.
PCR has also proved difficult to apply to samples which are old, which have been chemically treated (for example by fixing or embedding) or which are otherwise distressed. This significantly impairs the utility of the technique in for example the analysis of fixed archival material or of blood and tissue samples which are not fresh or which have not been stored under refrigeration. Fixation techniques now generally used do not permit ready release of DNA suitable for the subsequent amplification using conventional techniques, and whilst improved, less damaging, fixation techniques are being developed, the situation is not entirely satisfactory. Complicated treatment procedures, e.g. deparaffinization of paraffin-embedded material, proteinase digestion etc are generally required and in many cases amplification cannot be achieved at all. This applies also in the case of aged or non-refrigerated samples.
There therefore exists a need for an improved method for preparing nucleic acid for use in amplification procedures, which is quick and simple to perform and which may be used on aged, non-refrigerated, fixed or otherwise treated or distressed samples. The present invention addresses this need.
We have now found that nucleic acid may be isolated from a sample in a form directly suitable for amplification by a simple and easy to perform procedure which involves boiling or heating the sample to a high temperature and allowing it to cool, and depositing the nucleic acid onto a solid support. This procedure avoids many of the complicated and time-consuming treatment steps of the prior art and, more importantly, can successfully be directly applied to clinical and other blood-containing samples and to samples which are aged, non-refrigerated or fixed, where previous techniques have proved unsuccessful. More particularly, the invention is based on the surprising discovery that when a

REFERENCES:
patent: 4336173 (1982-06-01), Ugelstad
patent: 5200314 (1993-04-01), Urdea
patent: 5646001 (1997-07-01), Terstappen et al.
Chen and Viola, A Method to Detect ras Point Mutations in Small Subpopulations of Cells, Analytical Biochemistry 195:51-56, 1991.
Haliassos et al., Modification of Enzymatically Amplified DNA for the Detection of Point Mutations, Nucleic Acid Research 17:3606, 1989.
Kahn et al., Rapid and Sensitive Nonradioactive Detection of Mutant K-ras Genes Via `Enriched ` PCR Amplification, Oncogene 6:1079-1083, 1991.
Kahn et al., Rapid Nonradioactive Detection of ras Oncogenes in Human Tumors, Amplifictions 4:22-26, 1990.
Kumar and Barbacis, Oncogene Detection at the Single Cell Level, Oncogene 3:647-651, 1988.
Wahlberg et al., Rapid Detection and Sequencing of Specific in vitro Amplified DNA Sequences Using Solid Phase Methods, Molecular and Cellular Probes 4:285-297, 1990.
Technical Handbook, Molecular Biology, Dynal, pp. 1-48, 1992.

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