PCR KIT FOR CAPILLARY ELECTROPHORESIS
The present invention relates to a PCR kit for improving detection of particular nucleic acid motifs and for increasing the reliability of the results obtained. More precisely, the invention proposes to use modified primers having particular characteristics making it possible, during electrophoresis treatment, to detect, on samples containing nucleic acids, the equivalent of a characteristic encoding of particular nucleic acid motifs, this encoding being such that it makes it possible to better detect the presence of these motifs. The invention also proposes a method for detecting nucleic acid motifs of interest (for example STRs, mini-STRs or VNTRs) using such primers. It advantageously finds—but is not limited to—application in the field of capillary electrophoresis.
1 . PCR kit containing at least two primer pairs specific to a motif of interest to be identified on a nucleic acid, said pairs being adapted to generate, for said motif, at least two amplicons differentiable by their size and whose size differs by one base pair, by two base pairs, by three base pairs, or by four base pairs.
2 . PCR kit according to claim 1 , wherein said primer pairs are adapted to generate, for each motif, amplicons carrying a different label.
3 . Kit according to claim 1 , containing:
a reference primer pair adapted to specifically amplify motif A, producing an amplicon of size T (T base pairs), labeled with a label M, and
a modified primer pair adapted to specifically amplify motif A, producing an amplicon of size T′ different from T by one, by two, by three or by four base pairs more or fewer, labeled with a label M′ different from M.
4 . Kit according to claim 1 , containing, for each motif of interest to be identified on a nucleic acid, three primer pairs specific to said motif, said pairs being adapted to generate at least three amplicons differentiable by their size and/or their labeling.
5 . Kit according to claim 1 , containing a reference primer pair capable of specifically amplifying motif A, producing an amplicon of size T (T base pairs), labeled with a label M, and at least two other primer pairs selected from:
a modified primer pair adapted to specifically amplify motif A, producing an amplicon having a size T′ different from T by one, by two, by three or by four base pairs more or fewer, said amplicon being labeled with a label M,
a modified primer pair adapted to specifically amplify motif A, producing an amplicon having a size T, said amplicon being labeled with a label M′ different from M, and
a modified primer pair adapted to specifically amplify motif A, producing an amplicon having a size T′ different from T by one, by two, by three or by four base pairs more or fewer, said amplicon being labeled with a label M′ different from M.
6 . Method for detecting at least one nucleic acid motif of interest in a biological sample, wherein it uses a kit according to claim 1 and in that said nucleic acid motif of interest is detected when, for said motif, at least one amplicon per primer pair used specific of said motif is detected.
7 . Method according to claim 6 , comprising the following steps:
a) Obtaining a biological sample containing nucleic acids,
b) Contacting the biological sample with the at least two primer pairs and reagents required to amplify said nucleic acids,
c) Amplifying said nucleic acids under suitable conditions,
d) Detecting the amplicons obtained by means of their size and/or their labeling,
e) Generating a genetic profile according to the amplicons detected in step d).
8 . Method according to claim 6 , wherein said amplicons are detected by means of capillary electrophoresis.
9 . Method according to claim 6 , wherein said motif of interest is an STR.
10 . Method according to claim 6 , wherein, to detect said nucleic motif of interest, all the combinations of n peaks detected in the sample for each set of n peaks obtained are compared with the expected sets of n peaks contained in a list compiled beforehand.
11 . Method according to claim 6 , wherein, to detect said nucleic motif of interest, the following steps are carried out:
a) detecting all the valid sets of n peaks in the sample tested,
b) attributing each valid set of n peaks to a motif of interest sought.
12 . Method according to claim 7 wherein the steps of detecting and generating a genetic profile, d) and e), and if need be the steps of amplifying and contacting, are carried out automatically.