Size marker and method for controlling the resolution of an electropherogram
View Patent ↗The invention concerns a size marker for electrophoresis, characterized in that it contains several pairs of molecules the sizes of which are selected to allow the generation of a succession of double-peaks in one same electropherogram, said double-peaks being spaced apart two by two by a distance greater than the distance separating the two peaks of a double-peak. The invention also concerns a method for controlling the resolution of an electropherogram produced with a said marker.
1. A size marker for electrophoresis, characterized in that it contains several pairs of molecules the sizes of which are selected to allow the generation of a succession of only double-peaks in one same electropherogram, said double-peaks being spaced apart two by two by a distance greater than the distance separating the two peaks of a doublepeak, and characterized in that the size difference (“b”) between pairs of molecules contained in said marker and which correspond to successive double-peaks is greater than twice the size difference (“a”) between two molecules of one same pair (b>2a).
2. The marker according to claim 1 , wherein said molecules are molecules of DNA or RNA, and characterized in that the size difference (“a”) between two molecules of one same pair is between 1 and 6 base pairs.
3. The marker according to claim 2 , wherein “a” equals 1 base pair.
4. The marker according to claim 1 , wherein said molecules are molecules of DNA or RNA, and characterized in that “b” is equal to between 20 and 60 base pairs.
5. The marker according to claim 4 , wherein “b” equals 32 base pairs.
6. The marker according to claim 1 , characterized in that it contains at least three pairs of molecules, each adapted to generate double-peaks in one same electropherogram.
7. The marker according to claim 1 , wherein said molecules are chosen among: single or double strand DNA, RNA, polypeptides, proteins, organic medicinal products, inorganic anions and cations.
8. The marker according to claim 1 , characterized in that it comprises pairs of molecules which generate double-peaks in different regions of the electropherogram which correspond to different size differences “a”.
9. The marker according to claim 1 , characterized in that it comprises pairs of molecules which generate double-peaks in different regions of the electropherogram which correspond to constant size differences “a”.
10. The marker according to claim 1 , characterized in that the size difference (“b”) between the pairs of molecules generating successive double-peaks is identical for the whole marker, the double-peaks being regularly spaced along the electropherogram.
11. The marker according to claim 1 , characterized in that the size difference (“b”) between pairs of molecules generating successive double-peaks is variable.
12. A method for controlling the resolution of an electropherogram, characterized in that a signal corresponding to said electropherogram and/or data corresponding thereto are processed to detect the presence of double-peaks corresponding to the pairs of molecules of a marker according to claim 1 .
13. The method according to claim 12 wherein, to detect the presence of a double-peak, the ratio between the following is compared with a threshold:
the difference in height between the smallest peak of a double-peak and the valley of this double-peak; and
three times the value of the standard deviation (σ) of the height of all the valleys of the baseline signal from the preceding double-peak to the following double-peak.
14. The method according to claim 13 , characterized in that the curve of this ratio is superimposed over a corresponding electropherogram.
15. The method according to claim 12 , wherein, to detect the presence of a double-peak, the slope changes of the electropherogram signal are detected and it is detected that its slope becomes negative after a first maximum, passes through zero and re-becomes positive to reach a second maximum.
16. The method according to claim 12 , wherein, to detect the presence of a double-peak, comparative processing is performed whereby the ratio between the height of the valley between two peaks and the height of the smallest of the two peaks is compared with a threshold.
17. A computer programme product adapted to implement the steps of the method according to claim 12 when it is run on a computer.
18. An electrophoresis method wherein a sample to be analysed is subjected to migration by electrophoresis during which at least one detector generates an electropherogram signal, characterized in that:
at least one sample subjected to migration by electrophoresis contains a size marker according to claim 1 ,
and in that
the electropherogram signal and/or data corresponding thereto are processed to detect the presence of double-peaks corresponding to the pairs of molecules of said marker.
19. The capillary electrophoresis method according to claim 18 , characterized in that it is verified whether the detection of the double-peaks corresponding to the pairs of molecules of said marker is or is not satisfactory, and a capillary is changed as a function of the result of this verification or of prior verifications.
20. The electrophoresis method according to claim 18 , characterized in that the processing of the electropherogram signal and/or of data corresponding thereto to detect the presence of the double-peaks corresponding to the pairs of molecules of said marker is performed in real-time or a posteriori.
21. The electrophoresis method according to claim 18 , characterized in that it is performed to detect allelic variations between individuals as part of forensic analysis.