METHOD FOR DETERMINING THE QUANTITY OF A DNA FRAGMENT OF INTEREST BY ENZYMATIC AMPLIFICATION OF DNA

摘要

The present invention relates to a method for determining the quantity of a DNA fragment of interest in a sample to be analysed by enzymatic amplification in vitro of said fragment, characterized in that: 1) their is added to the sample to be analysed containing the DNA fragment of interest a standard DNA fragment different from the DNA fragment of interest but which may be amplified by the same primer oligonucleotides, the standard DNA fragment and the DNA fragment of interest differing in sequence and/or in size by not more than 10 %, and preferably not more than 5 nucleotides per strand; 2) the DNA fragment of interest and the standard DNA fragment are co-amplified with the same primers, preferably up to saturation of amplification of the DNA fragment of interest; 3) there is added to the reaction medium obtained in step 2): either two types of specific marked probe nucleotides, each respectively of the DNA fragments of interest and standard, and differing from the amplification primer oligonucleotides of step 2), or one or a plurality of marked primer oligonucleotides specific to the interest and standard DNA fragments and different from the primers of step 2), and one or more additional amplification cycles are carried out with the primer or primers so that, after denaturation of DNA, the primer or primers are hybridized with said fragment in a site appropriate for an extension by DNA polymerase generates marked DNA fragments having different sizes and/or sequences or with different markers whether they originate from DNA fragments of interest or standard fragments respectively, and 4) the initial quantity of DNA fragment of interest is determined as being the product of the initial quantity of standard DNA fragment and the ratio between the quantity of interest DNA fragment amplified and the quantity of standard DNA fragment amplified, ratio which is similar to that of quantities of marked DNA fragments originating respectively from amplified interest and standard DNA fragments obtained in step 3).