INTERNAL AMPLIFICATION CONTROL

摘要

Molecular biology techniques are widely used in genotyping applications and other areas such as biological research, forensic and diagnostic applications, including human identification and paternity testing and for diagnosis of infectious diseases or chimera analysis after allogeneic bone marrow transplantation as well the detection of genetic diseases and cancer. The most commonly used technique is the polymerase chain reaction (PCR) that allows the researchers to amplify the desired DNA requiring only tiny amounts of sample. Such amplification reactions are technically challenging and are often hampered by several practical issues such as the presence of PCR inhibitors, sample degradation and low quantities of said sample.;The invention addresses these issues by means of an internal amplification control consisting of a set of primers and an artificial template. The primers define two fragments of different sizes. The examples show that this system allows the researcher to distinguish between the presence of inhibitors and sample degradation.

主权项

1. Method for evaluating the amplification efficiency and/or the presence of inhibitors and/or degradation in an amplification reaction comprising a reaction mixture comprising
i. one or more internal nucleic acid templates with a length of between 50 and 2000 nucleotides, ii. at least 3 primers or at least 2 pairs of primers, which allow the generation of a first internal amplification product of between 20 and 800 nucleotides and at least one further internal amplification product of between 70 and 2000 nucleotides, wherein the 3 primers or 2 pairs of primers are specific for the one or more internal nucleic acid template and the first and second internal amplification product are amplification products of the one or more internal nucleic acid template, wherein the first internal amplification product is smaller than the second internal amplification product, iii. a sample nucleic acid to be analysed and primers specific for said nucleic acid and reagents for said amplification,wherein an amplification reaction is performed with said reaction mixture and the ratio of the smaller internal control amplification product to the larger internal control amplification product is analysed.