PCR controls
AssigneeOslo Universitetssykehus Hf
InventorsGuro E. Lind, Marine Jeanmougin, Heidi D. Pharo, Kim Andresen, Ragnhild Lothe
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Abstract
The present invention provides a method of quantification of a target nucleic acid, using at least any two of the genes SYT10, EPHA3, PLEKHF1 and KBTBD4 as control genes. In particular, the combination of the genes SYT10, EPHA3, PLEKHF1 and KBTBD4, known as the 4Plex, is provided as a control for nucleic acid quantification. The 4Plex has particular utility as a control for nucleic acid quantification by methylation-specific droplet digital PCR.
Claims21
A method of quantification of a target nucleic acid, wherein at least any two of the genes SYT10, EPHA3, PLEKHF1 and KBTBD4 are used as control genes, said method comprising:
- (i) amplifying the target nucleic acid, or a target region thereof, to yield a target amplicon, wherein the amplification is performed using a quantitative amplification method which allows absolute quantification and which uses primers;
- (ii) amplifying a target region of at least two of the control genes SYT10, EPHA3, PLEKHFI and KBTBD4, to yield a control gene amplicon for each of the at least two control genes, wherein the amplification is performed using a quantitative amplification method which allows absolute quantification and which uses primers, wherein no more than one target region of any one of the control genes is amplified;
- (iii) normalising the results of the target amplification of (i) using the results of the control amplification of the target regions of the at least two control genes of (ii);
- and
- (iv) based on (iii), determining a value for the amount of the target nucleic acid.
A kit comprising two or more primer sets suitable for use in PCR to amplify a target region within a target gene to generate an amplicon, said two or more primer sets selected from primer sets comprising:
- (i) a first primer and a second primer which bind within SYT10;
- (ii) a first primer and a second primer which bind within EPHA3;
- (iii) a first primer and a second primer which bind within PLEKHFI;
- and
- (iv) a first primer and a second primer which bind within KBTBD4;
- wherein the first primers and the second primers of the two or more primer sets each bind a site within the target gene which does not contain any CpG dinucleotides;
- and each primer set further comprises a fluorescent probe which binds the amplicon generated by PCR using the first primer and second primer of the primer set, wherein the fluorescent probe binds the amplicon at a site which does not contain any CpG dinucleotides.
Dependencies are inferred from the claim text.