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US 7,604,964 B2Granted
Method of determining the copy number of a nucleotide sequence
AssigneeMultigen Holding S.A.
InventorAndrea Cossarizza
Life of this patent
Estimating the term…
Abstract
The invention relates to a method of determining of accurately determining the copy number of a nucleotide sequence I in a sample using an amplification technique, such as PCR. In addition, a second nucleotide sequence II is also measured and calibration curves for each are made, from which the relative copy number CN can be determined. According to the present invention, accuracy is improved by performing multiple amplifications in a single well using real time PCR.
Claims28
A method of determining the relative copy number (CN) of a first nucleotide sequence I (NucSeqI) in a test sample using an amplification technique, said method comprising the steps of:
- (1) adding to the test sample that comprises NucSeqI and a chromosome-derived second nucleotide sequence II (NucSeqII), the following ingredients:
- nucleotides,
- primers,
- polymerase,
- a first probe specific to NucSeqI comprising a first fluorophore and a quencher, and/or a second probe specific to NucSeqII comprising a second fluorophore and a quencher wherein the first fluorophore and the second fluorophore are different;
- and optionally
- any additional reagents required for amplification,
- (2) carrying out the following amplification steps in one or more amplification cycles:
- (a) amplifying NucSeqI in said test sample,
- (b) amplifying NucSeqII in said test sample,
- (c) in a control sample, to which said ingredients of (1) are added, amplifying at multiple dilutions a third nucleotide sequence I′ (NucSeqI′) corresponding to NucSeqI to which said first probe is also specific, in the presence of said first probe,
- wherein the relationship of NucSeqI and NucSeqI′ is defined as
- (A) NucSeqI hybridizes to the complement of NucSeqI′, and
- (B) NucSeqI′ hybridizes to the complement of NucSeqI, both under stringent hybridization conditions, and, if NucSeqI and NucSeqI′ differ in length, the shorter of the two is at most 30% shorter than the other;
- and
- (d) in a control sample, to which said ingredients of (1) are added, amplifying at multiple dilutions a fourth nucleotide sequence II′ (NucSeqII′) corresponding to NucSeqII to which said second probe is also specific, in the presence of said second probe,
- wherein the relationship of NucSeqII and NucSeqII′ is defined as
- (A) NucSeqII hybridizes to the complement of NucSeqII′, and
- (B) NucSeqII′ hybridizes to the complement of NucSeqII, both under stringent hybridization conditions, and, if NucSeqII and NucSeqII′ differ in length, the shorter of the two is, at most, 30% shorter than the other;
- wherein
- (i) NucSeqI′ and NucSeqII′ are both localized on a single vector in which the ratio of NucSeqI′ to NucSeqII′ is known,
- (ii) standard curves SCI and SCII comprising at least two reference points are generated by amplification of NucSeqI′ and NucSeqII′, respectively, at multiple dilutions, wherein the starting quantity, concentration or dilution of NucSeqI′ and NucSeqII′ is known, and
- (iii) at least one pair of amplification reactions (a) and (b) or (c) and (d) is performed in a single container and monitored by fluorescence during amplification;
- (3) determining the results of the amplifications of step (2) expressed as threshold cycle (Ct) as a function of said starting quantity, concentration or dilution;
- (4) obtaining from the results in step (3) the following values:
- (i) “Conc-ISCI” which is the concentration, quantity or dilution in the test sample of NucSeqI determined from standard curve SCI;
- and
- (ii) “Conc-IISCII” which is the concentration, quantity or dilution in the test sample of NucSeqII determined from standard curve SCII,
- which standard curves express threshold cycle as a function of said starting concentration, quantity or dilution;
- and
- (5) determining from the values obtained in step (4) the relative CN of NucSeqI with respect to NucSeqII by the formula:
- Relative
-
-
- CN
- =
- Conc
- -
- I
- SCI
- Conc
- -
- II
- SCII
- thereby determining the relative CN of NucSeqI in said test sample.
42 more elements
A method of determining the relative CN of a first nucleotide sequence I (NucSeqI) in a test sample using an amplification technique, said method comprising the steps of:
- (1) adding to the test sample that comprises NucSeqI and a second nucleotide sequence II (NucSeqII), the following ingredients:
- nucleotides,
- primers,
- polymerase,
- a first probe specific to NucSeqI comprising a first fluorophore and a quencher, and/or a second probe specific to NucSeqII comprising a second fluorophore and a quencher, wherein the first fluorophore and the second fluorophore are different;
- and optionally
- any additional reagents required for amplification,
- (2) carrying out the following amplification steps in one or more amplification cycles:
- (a)amplifying NucSeqI in said test sample,
- (b)amplifying NucSeqII in said test sample,
- (c)in a control sample, to which said ingredients of (1) are added, amplifying at multiple dilutions a third nucleotide sequence I′ (NucSeqI′) corresponding to NucSeqI to which said first probe is also specific in the presence of said first probe,
- wherein the relationship of NucSeqI and NucSeqI′ is defined as
- (A) NucSeqI hybridizes to the complement of NucSeqI′, and
- (B) NucSeqI′ hybridizes to the complement of NucSeqI, both under stringent hybridization conditions, and, if NucSeqI and NucSeqI′ differ in length, the shorter of the two is at most 30% shorter than the other;
- and
- (d) in a control sample, to which said ingredients of (1) are added, amplifying at multiple dilutions a fourth nucleotide sequence II′ (NucSeqII′) corresponding to NucSeqII to which said second probe is also specific, in the presence of said second probe,
- wherein the relationship of NucSeqII and NucSeqII′ is defined as
- (A) NucSeqII hybridizes to the complement of NucSeqII′, and
- (B) NucSeqII′ hybridizes to the complement of NucSeqII, both under stringent hybridization conditions, and, if NucSeqII and NucSeqII′ differ in length, the shorter of the two is, at most, 30% shorter than the other;
- wherein
- (i) NucSeqI′ and NucSeqII′ are both localized on a single vector in which the ratio of NucSeqI′ to NucSeqII′ is known,
- (ii) standard curves SCI and SCII comprising at least two reference points are generated by amplification of NucSeqI′ and NucSeqII′, respectively, at multiple dilutions, wherein the starting quantity, concentration or dilution of NucSeqI′ and NucSeqII′ is known, and
- (iii) at least one pair of amplification reactions (a) and (b) or (c) and (d) is performed in a single container and monitored by fluorescence during amplification;
- (3) determining the results of the amplifications of step (2) expressed as threshold cycle (Ct) as a function of said starting quantity, concentration or dilution;
- (4) obtaining from the results in step (3) the following values:
- (i) “Conc-ISCI” which is the concentration quantity or dilution in the test sample of NucSeqI determined from standard curve SCI;
- and
- (ii) “Conc-ISCI” which is the concentration quantity or dilution in the test sample of NucSeqII determined from standard curve SCII, which standard curves express threshold cycle as a function of said starting concentration quantity or dilution;
- and
- (5) determining from the values obtained in step (4) the relative CN of NucSeqI with respect to NucSeqII by the formula:
- Relative
-
-
- CN
- =
- Conc
- -
- I
- SCI
- Conc
- -
- II
- SCII
- thereby determining the relative CN of NucSeqI in said test sample.
41 more elements
Dependencies are inferred from the claim text.