# US 8,541,206 B2 · Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids

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- Number: US 8,541,206 B2
- Document ID: US8541206B2
- Status: Granted
- Kind: B2
- Application number: 12/086,897
- Filed: 2006-12-28
- Published: 2011-01-06
- Granted: 2013-09-24
- Earliest claimed: 2006-12-28
- Assignees: Institut Pasteur
- Applicants: Simon Wain-Hobson; Jean-Pierre Vartanian
- Inventors: Simon Wain-Hobson; Jean-Pierre Vartanian
- Web page: https://patentgrep.com/patent/US8541206B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6827: for detection of mutation or polymorphism
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for enriching a mutant nucleic acid in a mixture of nucleic acids, wherein the method comprises: (a) providing a nucleic acid mixture comprising a parental nucleic acid and a mutant nucleic acid of the parental nucleic acid; and (b) amplifying the nucleic acids in the nucleic acid mixture by polymerase chain reaction (PCR); wherein the mutant nucleic acid is a G→A mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence to form an AT-rich nucleic acid variant of the parental nucleic acid; and wherein the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental nucleic acid to thereby enrich the mutant nucleic acid in the nucleic acid mixture.

## Claims

### Claim 1

A method for the differential amplification of a nucleic acid by polymerization chain reaction (PCR), wherein the method consists essentially of:
(a) providing a nucleic acid mixture comprising a parental DNA and a GC allele of the parental DNA;
(b) carrying out PCR in the nucleic acid mixture in a reaction medium containing deoxyinosine (dITP), or in a reaction medium containing deoxy 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP, wherein Guanine bases (G) are converted into Inosine bases (I) and Adenosine bases (A) are converted into 2,6-diaminopurine bases (D); and
(c) subjecting nucleic acids of (b) to PCR using a denaturation temperature 1-10° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental DNA to thereby selectively amplify the GC allele in the nucleic acid mixture.

### Claim 2 (depends on 1)

The method as claimed in claim 1, wherein the denaturation temperature is 1-6° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 3 (depends on 1)

The method as claimed in claim 1, wherein the denaturation temperature is 1-3° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 4 (depends on 1)

The method as claimed in claim 1, which further comprises detection of one or more of the nucleic acid products of (c).

### Claim 5 (depends on 1)

The method as claimed in claim 1, wherein differential amplification is carried out using about equimolar ratio of dTTP, dCTP, dDTP, and dITP.

### Claim 6 (depends on 1)

The method as claimed in claim 1, wherein amplifications are carried out with a thermostable DNA polymerase consisting of Taq polymerase.

### Claim 7 (depends on 1)

The method as claimed in claim 1, wherein the mixture of nucleic acids is devoid of said parental DNA corresponding to the GC alleleic DNA to be amplified.

### Claim 8 (depends on 1)

The method as claimed in claim 1, wherein standard amplification is carried out in the following buffer conditions 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 200 μM of each primer and 5U of Taq DNA polymerase or a variant thereof in a final volume of 50 μl.

### Claim 9 (depends on 1)

The method as claimed in claim 1, wherein the differential amplification is carried out in PCR conditions enabling temperature gradients to be generated in denaturation temperature, in order to enable amplification of the GC alleleic DNA selectively with respect to amplification of the corresponding parental nucleic acid.

### Claim 10 (depends on 9)

The method as claimed in claim 9, wherein the temperature gradient for differential amplification is suitable to determine the minimum denaturation temperature for the parental nucleic acid.

### Claim 11 (depends on 1)

The method as claimed in claim 1, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 15° C.

### Claim 12 (depends on 1)

The method as claimed in claim 1, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 10° C.

### Claim 13 (depends on 1)

The method as claimed in claim 1, wherein the primers used for PCR in differential amplification are devoid of A and T nucleobases in the 1 to 6 nucleotides of their 3′ end.

### Claim 14 (depends on 1)

The method as claimed in claim 1, wherein the GC alleleic nucleic acid to be enriched is originating from a pathogenic organism or from a gene associated with a pathogenic condition in a host.

### Claim 15 (depends on 14)

The method as claimed in claim 14, wherein the pathogenic organism is a DNA virus or a RNA virus.

### Claim 16 (depends on 15)

The method as claimed in claim 15, wherein the virus is HIV, MV, or a RVFV.

### Claim 17 (depends on 14)

The method as claimed in claim 14, wherein the gene associated with a pathogenic condition in a host relates to a tumor state.

### Claim 18 (depends on 1)

The method as claimed in claim 1, wherein the CG alleleic nucleic acid is a A→G, a A→C, a T→G or a T→C allele, variant or mutant of the parental nucleic acid, which pairs with a fully complementary nucleic acid sequence.

### Claim 19 (depends on 1)

The method as claimed in claim 1, wherein the CG alleleic nucleic acid variant is a small deletion mutant of the parental nucleic acid.

### Claim 20 (depends on 19)

The method as claimed in claim 19, wherein the deletion comprises 1 or 2 bp.

### Claim 21 (depends on 1)

The method as claimed in claim 1, wherein the products of the PCR are detected by gel filtration or ion-exchange chromatography.

### Claim 22 (depends on 1)

The method as claimed in claim 1, wherein products of the PCR are identified by relative location in the gel.

### Claim 23 (depends on 1)

The method as claimed in claim 1, wherein the modified bases are dITP or dDTP.

### Claim 24 (depends on 1)

The method as claimed in claim 1, wherein step b) of amplification of nucleic acids by PCR is carried out with non-standard PCR buffer comprising tetraethyl-ammonium chloride, methanol, or polyethylene glycol.

### Claim 25 (depends on 1)

The method as claimed in claim 1, wherein the parental nucleic acid comprises 40 to 500 bases.

### Claim 26 (depends on 1)

The method as claimed in claim 1, wherein the parental nucleic acid comprises 40 to 80 bases.

### Claim 27 (depends on 1)

The method as claimed in claim 1, wherein the window of observation is between 10-30 bases.

### Claim 28

A method of differential DNA denaturation PCR (3DI-PCR), wherein the method consists essentially of:
(a) providing a nucleic acid mixture comprising a parental DNA having up to about 500 bp and a GC allele of the parental DNA having up to about 500 bp;
(b) carrying out PCR in the nucleic acid mixture in a reaction medium containing deoxyinosine (dITP), deoxy 2,6-diaminopurine triphosphate (dDTP), or dITP and dDTP, wherein Guanine bases (G) are converted into Inosine bases (I) and Adenosine bases (A) are converted into 2,6-diaminopurine bases (D); and
(c) subjecting nucleic acids of (b) to PCR using primers having about 20-25 bp at a denaturation temperature 1-10° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental nucleic acid, to thereby selectively amplify a region of about 20-80 bp of the GC allele of the parent DNA in the nucleic acid mixture.

### Claim 29 (depends on 28)

The method as claimed in claim 28, wherein the denaturation temperature is 1-6° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 30 (depends on 28)

The method as claimed in claim 28, wherein the denaturation temperature is 1-3° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 31 (depends on 28)

The method as claimed in claim 28, which further comprises detection of one or more of the nucleic acid products of (c).

### Claim 32 (depends on 28)

The method as claimed in claim 28, wherein differential amplification is carried out using about equimolar ratio of dTTP, dCTP, dDTP, and dITP.

### Claim 33 (depends on 28)

The method as claimed in claim 28, wherein the differential amplification is carried out in PCR conditions enabling temperature gradients to be generated in denaturation temperature, in order to enable amplification of the GC alleleic nucleic acid selectively with respect to amplification of the corresponding parental nucleic acid.

### Claim 34 (depends on 28)

The method as claimed in claim 28, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 15° C.

### Claim 35 (depends on 28)

The method as claimed in claim 28, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 10° C.

### Claim 36 (depends on 28)

The method as claimed in claim 28, wherein the primers used for PCR in differential amplification are devoid of A and T nucleobases in the 1 to 6 nucleotides of their 3 end.

### Claim 37 (depends on 28)

The method as claimed in claim 28, wherein the products of the PCR are detected by gel filtration or ion-exchange chromatography.

### Claim 38 (depends on 28)

The method as claimed in claim 28, wherein products of the PCR are identified by relative location in the gel.

### Claim 39 (depends on 28)

The method as claimed in claim 28, wherein the modified bases are dITP or dDTP.

### Claim 40

A method of inverse differential DNA denaturation PCR (3DI-PCR), wherein the method consists essentially of:
(a) providing a nucleic acid mixture comprising a parental DNA and a mutant DNA of the parental DNA containing 2-5 GC alleleic sites;
(b) carrying out PCR in the nucleic acid mixture in a reaction medium containing deoxyinosine (dITP), deoxy 2,6-diaminopurine triphosphate (dDTP), or dITP and dDTP, wherein Guanine bases (G) are converted into Inosine bases (I) and Adenosine bases (A) are converted into 2,6-diaminopurine bases (D); and
(c) subjecting nucleic acids of (b) to PCR using primers having about 20-25 bp at a denaturation temperature 1-10° C. lower than the lowest denaturation temperature (Tp) that allows amplification of the parental nucleic acid, to thereby produce a differentially amplified PCR fragment of 60-80 bp of the GC allele of the parent nucleic acid in the nucleic acid mixture.

### Claim 41 (depends on 40)

The method as claimed in claim 40, wherein the denaturation temperature is 1-6° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 42 (depends on 40)

The method as claimed in claim 40, wherein the denaturation temperature is 1-3° C. lower than the lowest Tp that allows amplification of the parental nucleic acid.

### Claim 43 (depends on 40)

The method as claimed in claim 40, which further comprises detection of one or more of the nucleic acid products of (c).

### Claim 44 (depends on 40)

The method as claimed in claim 40, wherein differential amplification is carried out using about equimolar ratio of dTTP, dCTP, dDTP, and dITP.

### Claim 45 (depends on 40)

The method as claimed in claim 40, wherein the products of the PCR are detected by gel filtration or ion-exchange chromatography.

### Claim 46 (depends on 40)

The method as claimed in claim 40, wherein the differential amplification is carried out in PCR conditions enabling temperature gradients to be generated in denaturation temperature, in order to enable amplification of the GC-rich mutant nucleic acid selectively with respect to amplification of the corresponding parental nucleic acid.

### Claim 47 (depends on 46)

The method as claimed in claim 46, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 15° C.

### Claim 48 (depends on 47)

The method as claimed in claim 47, wherein the differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 10° C.

### Claim 49 (depends on 40)

The method as claimed in claim 40, wherein the primers used for PCR in differential amplification are devoid of A and T nucleobases in the 1 to 6 nucleotides of their 3′ end.

### Claim 50 (depends on 40)

The method as claimed in claim 40, wherein the denaturation temperature is 1-3° C. lower than the lowest Tp that allows amplification of the parental nucleic acid, differential amplification is carried out using about equimolar ratio of dTTP, dCTP, dDTP, and dITP, and differential amplification is carried out in a denaturation temperature gradient in the range of 1 to 10° C., and the primers used for PCR in differential amplification are devoid of A and T nucleobases in the 1 to 6 nucleotides of their 3′ end.

### Claim 51 (depends on 50)

The method as claimed in claim 50, which further comprises detection of one or more of the nucleic acid products of (c).

### Claim 52 (depends on 50)

The method as claimed in claim 50, wherein the mutant nucleic acid in (a) contains 3 or 4 GC alleleic sites.

### Claim 53 (depends on 50)

The method as claimed in claim 50, wherein the mutant nucleic acid in (a) contains two GC alleleic sites.

### Claim 54 (depends on 52)

The method as claimed in claim 52, wherein the denaturation temperature gradient is 63° C. to 72° C.

### Claim 55 (depends on 54)

The method as claimed in claim 54, wherein the mutant DNA contains about 2 alleleic sites per 265 bp.