US20070154892A1 - Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids - Google Patents

Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids Download PDF

Info

Publication number
US20070154892A1
US20070154892A1 US11/321,048 US32104805A US2007154892A1 US 20070154892 A1 US20070154892 A1 US 20070154892A1 US 32104805 A US32104805 A US 32104805A US 2007154892 A1 US2007154892 A1 US 2007154892A1
Authority
US
United States
Prior art keywords
nucleic acid
parental
mutant
pcr
mixture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/321,048
Other languages
English (en)
Inventor
Simon Wain-Hobson
Jean Vartanian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur de Lille
Original Assignee
Institut Pasteur de Lille
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut Pasteur de Lille filed Critical Institut Pasteur de Lille
Priority to US11/321,048 priority Critical patent/US20070154892A1/en
Assigned to INSTITUT PASTEUR reassignment INSTITUT PASTEUR ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VARTANIAN, JEAN-PIERRE, WAIN-HOBSON, SIMON
Priority to JP2008548048A priority patent/JP2009524412A/ja
Priority to CA002635109A priority patent/CA2635109A1/en
Priority to PCT/IB2006/004187 priority patent/WO2007091125A2/en
Priority to US12/086,897 priority patent/US8541206B2/en
Priority to EP06849524.1A priority patent/EP1979492B1/en
Publication of US20070154892A1 publication Critical patent/US20070154892A1/en
Priority to US12/691,377 priority patent/US20100184017A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to the differential amplification of mutant nucleic acids by polymerase chain reaction (PCR) in a mixture of nucleic acids. More particularly, this invention relates to preferentially amplifying copies of one or more mutant nucleic acids in a mixture of nucleic acids containing the parental nucleic acid of the mutant nucleic acids in order to enrich the mixture in copies of the mutant nucleic acids.
  • PCR polymerase chain reaction
  • One embodiment of this invention provides a method for enriching a mutant nucleic acid in a mixture of nucleic acids.
  • 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).
  • the mutant nucleic acid is an AT-rich nucleic acid variant of the parental nucleic acid.
  • the AT-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C.
  • the mutant nucleic acid is thereby enriched in the nucleic acid mixture.
  • the G ⁇ MA mutant of the parental nucleic acid pairs with a fully complementary nucleic acid sequence to form the AP-rich nucleic acid variant.
  • This invention also provides 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).
  • the mutant nucleic acid in this embodiment is a GC-rich nucleic acid variant of the parental nucleic acid.
  • the GC-rich nucleic acid variant is denatured and selectively amplified by carrying out PCR using a denaturation temperature 1-3° C.
  • PCR is carried out in a reaction medium containing deoxyinosine triphosphate (dITP), or in a reaction medium containing 2,6-diaminopurine triphosphate (dDTP), or in a reaction medium containing dITP and dDTP.
  • dITP deoxyinosine triphosphate
  • dDTP 2,6-diaminopurine triphosphate
  • the methods of the invention can include an optional step of detecting the products of the PCR.
  • the PCR can be carried out in the absence of the parental nucleic acid.
  • FIG. 1 depicts differential DNA denaturation amplification of G ⁇ A-hypermutated HIV-1 genomes.
  • (a) Four sequences harbouring 3, 8, 14 and 18 G ⁇ A transitions compared with the reference sequence (0) were amplified under standard PCR conditions with a denaturation temperature of 95° C. M and C denote molecular mass markers and negative control, respectively.
  • 293T/PBMC refers to material amplified from PBMCs infected by an HIV-1 ⁇ vif virus stock produced by transfection of 293T cells.
  • the same samples as in (a) were amplified with a denaturation temperature of 83° C.
  • FIG. 2 depicts a collection of G ⁇ A-hypermutated HIV-1 V1V2 region sequences derived from ⁇ vif stock virus grown on 293T cells [293T/PBMC, FIG. 1 ( a - c )]. For clarity, only a 189 bp region of the 304 bp segment that was amplified is shown. Sequences are aligned with respect to the parental sequence. Only differences are shown. Hyphens denote gaps. Clone designation is shown to the left. Analysis of material from the 95° C. amplification failed to identify any hypermutated genomes.
  • FIG. 3 depicts a collection of AT-rich poliovirus VP1 segments derived from a patient with post-vaccinal acute flaccid paralysis. For clarity, only a 109 bp region of the 480 bp segment that was amplified is shown. Sequences are aligned with respect to poliovirus Sabin 1. Only differences are shown. Clone designation is shown to the right. The 3D-PCR-amplified segments bore one to six GC AT transitions compared with Sabin 1. Analysis of material from the 95° C. amplification yielded two substitutions among 17 clones in the same sequence.
  • FIG. 4A depicts the sequences of two alleles of the p21 ras gene. Primers are underlined in the Figure.
  • FIG. 4B depicts primers for amplifying a 19 bp window of the p21 ras gene.
  • FIG. 5 depicts the chemical structure of dUTP and dCTP analogues that can optionally be substituted for dTTP.
  • A adenosine
  • T/U thymine/uracil
  • G guanosine
  • C cytidine
  • Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes.
  • use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies.
  • the latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T p ) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3° C. lower than T p .
  • T p the lowest denaturation temperature
  • differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3° C. lower than T p .
  • Application of this sensitive method to two different viruses made it possible to identify human immunodeficiency virus type 1 G ⁇ A hypermutants in a situation
  • method according to this invention allows differential amplification of DNA segments differing by one to many GC->AT transitions.
  • degree of substitution directly impacts the melting temperature of the DNA, the lower the denaturation temperature the more substituted the genomes amplified.
  • loci may have widely different base compositions the conditions can be optimized for each segment.
  • the method of the invention applies to DNA or cDNA (reverse transcribed RNA) no matter the origin.
  • Preferred sources of nucleic acids are HIV-1, HIV-2, poliovirus, and measle virus.
  • this invention relates to the amplification of segments of DNA by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the terms “polymerase chain reaction” and “PCR” are used in their conventional sense as an in vitro method for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in a target DNA.
  • a repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers.
  • thermostable DNA polymerase such as Taq polymerase isolated from Thermus aquaticus , makes it possible to carry out the PCR reaction of the invention in a simple and robust manner, which can be automated using a conventional thermal cycling device.
  • the PCR reaction of the invention can be carried out using conventional reaction components, such as, the template DNA, primers, Taq or another polymerase, dNTP's, and buffer.
  • the reaction can be carried out in the conventional manner by simply cycling the temperature within a reaction chamber.
  • the specificity and yield of the amplification reaction can be regulated by controlling well-known parameters, such as enzyme, primer, dNTP, and Mg ++ 0 concentrations, as well as the temperature cycling profile.
  • PCR is used according to the invention.
  • the amplification can be initially performed in a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments) using the “Step-Cycle” program and reagents recommended by the manufacturer. For any given pair of oligonucleotide primers an optimal set of conditions can then be established.
  • the lowest denaturation temperature that allows amplification of the parental sequence is determined.
  • This lowest denaturation temperature is termed T p .
  • Differential amplification of AT-rich nucleic acid variants is obtained by using the same reagents and the same step-cycle program, except that the denaturation temperature for each cycle of PCR is about 1 to 3° C., preferaby 1° C., lower than T p .
  • the temperature employed for the denaturation step of PCR will be a temperature at which the mutant nucleic acid is preferentially amplified relative to the parental nucleic acid.
  • Preferential amplification can be determined, for example, by gel electrophoresis or by direct sequencing of PCR products or by detection with a labeled probe.
  • the nucleic acid mixture employed in the methods of the invention can comprise a parental nucleic acid and at least one mutant nucleic acid of the parental nucleic acid.
  • the mutant nucleic acid can contain at least one G ⁇ A mutation relative to the parental nucleic acid to form, after base pairing, an AT-rich nucleic acid variant of the parental nucleic acid.
  • the mutant nucleic acid can contain at least one A ⁇ G mutation relative to the parental nucleic acid to form, after base pairing, a GC-rich nucleic acid variant of the parental nucleic acid.
  • the mixture can contain a mutant nucleic acid having both G ⁇ A and A ⁇ G mutations at different loci, or the mixture can contain two or more nucleic acid mutants each containing either one or more G ⁇ A mutations or one or more A ⁇ G mutations.
  • the number of mutations in the mutant nucleic acids is typically 1-18 mutations compared to the parental nucleic acid.
  • the methods of the invention apply to any DNA or cDNA (reverse transcribed RNA) fragment no matter the origin.
  • An important property of the PCR reaction of the invention, particularly in diagnostic applications, is the capacity to amplify a target sequence from crude DNA preparations as well as from degraded DNA templates.
  • the DNA in the sample to be amplified need not be chemically pure to serve as a template provided that the sample does not contain inhibitors of the polymerase.
  • the ability to amplify specific sequences from crude DNA samples has important implications for research applications, for medical diagnostic applications, and for forensics.
  • the primers used in PCR can contain mismatches relative to the sequences to which they base-pair.
  • the primers can be degenerate, as is described for primers SK122/SK123 used to hybridize with the V1V2 region of the HIV-1 envelope gene in the Examples hereinafter. If the primers contain mismatches relative to the sequence to which they base-pair, the hybridization step of PCR can be optimized independently of the denaturation step of PCR. In addition, it will be understood that the primers can contain mismatches relative to the parental sequence.
  • the length of the primers has not been found to be critical in carrying out the methods of the invention. Standard length primers can be employed, and optimal primer length can be determined by routine experimentation. Typically, the primers will be about 20-25 bp, but may be longer or shorter.
  • the length of the parental sequence has not been found to be critical in carrying out the methods of the invention.
  • the parental sequence will be up to about 500 bp. It will be understood that longer or shorter sequences can be employed.
  • mutant nucleic acids being amplified has not been found to be critical in the methods of the invention. Mutant nucleic acids up to about 500 bp can be employed, although it will frequently be more convenient to use shorter sequences.
  • the region of the mutant nucleic acids being amplified can vary depending upon the target nucleic acids.
  • the region amplified can comprise about 20, 30, 40, 50, or 60 bp, although longer or shorter sequences are contemplated by the invention. Amplified regions of 19 and 30 bp are described for p12 ras gene in the Examples hereinafter.
  • the amplified region may affect the manner in which the amplified nucleic acids are detected.
  • the window between two primers can be 3-12 nucleotides, but in this case, using 20 bp primers, the bands of the PCR products are about 43-52 base pairs. Nucleic acid molecules of this size can not readily be detected by electrophoresis in agarose gel, but they can be detected in polyacrylamide gel.
  • the detection method can be adapted to the characteristics of the amplified PCR product.
  • Preferred detection methods are gel electrophoresis in agarose or acrylamide gel, capillary electrophoresis, or chromatography, especially gel filtration or ion-exchange chromatography.
  • the methods of this invention have a wide variety of uses.
  • the methods can be employed to characterize the origin of parental DNA or to detect mutations characteristic of human gene disorders.
  • the methods can also be employed for detecting G ⁇ A mutant strains of HIV, particularly G ⁇ A hypermutants, that are resistant to anti-retroviral drugs.
  • the methods of the invention can be employed for detecting neurovirulent vaccine-derived poliovirus isolates that cause vaccine-associated paralytic poliomyelitis.
  • the methods of the invention are used to amplify parental and mutant nucleic acids from measle virus.
  • the methods of the invention are useful for detecting specific mutations at specific sites, which have previously been characterized and sequenced.
  • the methods of the invention are also useful for detecting the presence of unknown sequence differences in a given length of DNA.
  • the methods are useful for detecting known mutations, and are particularly useful for rapidly detecting multi-allelic loci. It will be understood that the methods of the invention can also be used to amplify sequences containing small deletions, such as 1or 2 bp deletions.
  • the method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA it is possible to quantitate the fraction of AT rich genomes within a sample.
  • Taqman PCR can be performed at 95° C. and the selective temperature to determine the copy number per sample. The ratio of the two values can give the relative concentration of AT-rich alleles with respect to the total concentration of all alleles.
  • the methods according to the invention encompass variants that use modified bases that can influence slightly the melting temperature of DNA.
  • dUTP can be used to replace dTTP ( FIG. 5 ).
  • modified derivatives of dCTP such as 5-methyl dCTP, 5-fluoro dCTP, 5-chloro dCTP, 5-bromo dCTP, or 5-iodo dCTP, which can be incorporated into DNA by Taq polymerase or another thermostable DNA polymerase ( FIG. 5 ).
  • dCTP analogues are particularly interesting, for when incorporated into DNA, the DNA melts to higher temperatures (Hoheisel et al., 1990, Wong et al., 1991). Accordingly, use of any one of these modified bases will enhance the discrimination between the parental sequence and AT rich allele.
  • the methods of the invention encompass the use of non-standard PCR buffer conditions, particularly the use of certain salts and salt concentrations and the use of organic molecules.
  • the denaturation temperature can be influenced by the nature of the ion and ionic strength, for example tetraethylammonium chloride (Muraoka et al., 1980) and the use of small organic molecules, such as methanol or polyethylene glycol, to cite just two (Muraoka et al., 1980, Votavova et al., 1986).
  • the methods of the invention can be used to detect small deletions in an allele.
  • the melting temperature is a function of the number of hydrogen bonds distinguishing two alleles. Deletion of a single base will remove 2 or 3 hydrogen bonds, and hence will melt to lower temperature. Larger deletions will be detected more readily.
  • the method of the invention can be used to selectively amplify alleles with small deletions, for example, mitochondrial DNA or microsatelites associated with a disease susceptibility gene, although these are mentioned as examples and not to limit the invention.
  • the method of the invention can be used in the search for single nucleotide polymorphisms (SNPs). Being temperature based, no allele specific oligonucleotides are necessary.
  • the method of the invention can detect any mutation provided that it reduces the melting temperature of the allele.
  • GC->AT substitutions represent the most frequent substitutions (40-50%) characterizing human gene disorders, p53 inactivating mutations or those of pseudogenes compared to the orthologous gene, it is possible to apply the method of the invention to the detection of a mutation characteristic of a pre-tumourous cell in a blood sample or in the characterization of human genotypes via SNP typing.
  • 3DPCR The methods of the invention, designated “3DPCR,” can be used to selectively amplify AT rich alleles from the normal counterpart. This follows on from the fact that an A:T base pair involves 2 hydrogen bonds, while a G:C pair involves 3 ( FIG. 1 ). Consequently, it has been demonstrated that the denaturation temperature of an AT rich allele will be slightly lower than that of the normal counterpart. The converse, the selective amplification of a G:C rich allele compared to a normal counterpart, is not amenable to analysis by 3DPCR because it would melt to a higher temperature than the normal allele, and at higher temperatures both the normal and GC rich alleles will be amplified.
  • dITP Deoxyinosine triphosphate
  • Inosine lacks the amino group at position 2 compared to guanosine.
  • dITP forms only 2 hydrogen bonds with dCTP ( FIG. 5 ).
  • G or I pairs with C the specificity of base pairing is preserved
  • D 2,6-diamino purine
  • D bears an additional amino group compared to adenosine (2-aminopurine).
  • it base pairs with thymidine via 3 hydrogen bonds ( FIG. 5 ). Again the information content is preserved (D or A pairs with T).
  • dDTP and dITP can substitute for dATP and dGTP in a PCR reaction, indeed both can be used in the same reaction, PCR material so derived will have the inverse melting properties compared to products bearing the canonical bases, dATP and dGTP.
  • G:C rich alleles, (I:C in fact, 2 hydrogen bonds) will melt to slightly lower temperatures than the normal A:T allele (D:T in fact, 3 hydrogen bonds).
  • performing 3DPCR of the invention with these modified bases allows selective amplification of A:T rich alleles.
  • the invention thus concerns the use of a lower melting temperature to amplify a subset of alleles that can be distinguished by a fractionally lower DNA melting temperature.
  • this invention also involves the use of dDTP and dITP as a means to convert a G:C rich allele into DNA that melts at a lower temperature.
  • This method of the invention is termed “inverse 3DPCR” or “i3DPCR” to emphasize that it allows amplification of G:C rich alleles as opposed to A:T rich alleles, which 3DPCR does.
  • the i3DPCR method according to the invention is well adapted to selectively amplifying such viral hypermutants.
  • the viral paradigm concerns measles virus hypermutants.
  • A->G hypermutants have been described for parainfluenza virus, respiratory syncytial virus, vesicular stomatitis virus, and some retroviruses including HIV.
  • the method of the invention is useful in basic research involving these and other diseases.
  • the i3DPCR method allows differential amplification of DNA segments differing by one to many AT->GC transitions. As the degree of substitution directly impacts the melting temperature of the DNA, the lower the denaturation temperature the more substituted the genomes amplified. As different loci may have widely different base compositions the conditions can be optimized for each segment.
  • the i3DPCR method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA, it is possible to quantitate the fraction of GC rich genomes within a sample.
  • Taqman PCR can be performed at 95° C. and the selective temperature to determine the copy number per sample. The ratio of the two values can give the relative concentration of GC-rich alleles with respect to the total concentration of all alleles.
  • the i3DPCR method encompasses variants that use modified bases that can influence slightly the melting temperature of DNA.
  • modified bases that can influence slightly the melting temperature of DNA.
  • 5-bromodUTP or dUTP can be used to replace dTTP.
  • the differences may be small. Accordingly, use of any one of these modified bases will enhance the discrimination between the parental sequence and GC-rich allele.
  • the i3DPCR methods cover the use of non-standard PCR buffer conditions, particularly the use of certain salts and salt concentrations and the use of organic molecules.
  • the denaturation temperature can be influenced by the nature of the ion and ionic strength, for example, tetraethylammonium chloride (Muraoka et al., 1980), and the use of small organic molecules, such as methanol or polyethylene glycol, to cite just two (Muraoka et al., 1980, Votavova et al., 1986).
  • i3DPCR can amplify GC-rich DNA from genomes.
  • G:C rich DNA is usually synonymous with coding regions and can help in identifying genes within genomes.
  • i3DPCR can be used to identify single point mutations in a small window.
  • 3DPCR has been used to identify a single G->A base change in a small locus of between 60-80 base pairs (bp).
  • i3PCR can identify a single A->G or T->C mutation in a small locus of 60-80 bp.
  • the i3DPCR method can be used to identify alleles with G/C rich point mutations within a mass of normal A:T alleles. The obvious example is to look for mutations characteristic of a pre-tumoral cell.
  • 3DPCR and i3DPCR can pick up 85% of all mutations characteristic of human gene disorders or p53 inactivating lesions (Krawczak et al., 1995, Li et al., 1984). The 15% of remaining mutations concern G ⁇ ->C and A ⁇ ->T transversions. While the number of base pairs is not altered by the mutations, it is possible that stacking energies will be affected, which could lead to a change in melting temperature. If so, then 3DPCR and i3DPCR can be used to identify these mutations too.
  • the human immunodeficiency virus (HIV) Vif protein intercepts the host-cell proteins APOBEC3F and APOBEC3G, preventing their incorporation into budding virions (Harris et al., 2003; Wiegand et al., 2004; Zheng et al., 2004).
  • the resulting Vif/APOBEC3 complexes are shunted to the proteasome for degradation (Sheehy et al., 2003; Yu et al., 2003).
  • APOBEC3 genes on human chromosome 22 at least five are transcribed (Jarmuz et al., 2002; http://genecards.bcgsc.bc.ca).
  • APOBEC1 specifically edits the apolipoprotein B mRNA in the environment of the intestine (Teng et al., 1993).
  • APOBEC3C, -3F and -3G are able to extensively deaminate single-stranded DNA (Harris et al., 2003; Lecossier et al., 2003; Suspene et al., 2004; Wiegand et al., 2004; Yu et al., 2004).
  • APOBEC3F and ⁇ 3G appear to be packaged into the virion (Harris et al., 2003; Bishop et al., 2004; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004). It is of note that APOBEC3F and -3G are packaged during budding from the donor cell and do not enter the replication complex of an incoming virion. Consequently, as soon as minus-strand viral cDNA is synthesized in the next round of infection, the numerous multiple C residues are deaminated, yielding U. Following plus-strand DNA synthesis, the U residues are copied into A, giving rise to so-called G ⁇ A hypermutants, by reference to the viral plus strand (Pathak & Temin, 1990; Vartanian et al., 1991).
  • FIG. 1 also shows nested PCR material (293T/PBMC) corresponding to the same V1V2 region amplified from peripheral blood mononuclear cells (PBMCs) that had been infected with a ⁇ vif derivative of HIV-1 pNL4.3 following transfection of 293T cells.
  • the denaturation temperature was 83° C.
  • the fact that this material represented differentially amplified G ⁇ A hypermutants was indicated when the 3D-PCR products were electrophoresed in a gel containing HA-yellow ( FIG. 1 c, 293T/PBMC).
  • FIG. 2 shows nested PCR material amplified at 95° C. identified only wild-type DNA (not shown).
  • the HIV-1 ⁇ vif virus stock was made by using the 293T cell line, which is widely used as not only can it be transfected easily, but also it is considered not to express APOBEC3 molecules.
  • the 293T cell line had become clonally heterogeneous, so that APOBEC3F [preference for 5′ TpC dinucleotide, GpA on viral plus strand (Harris et al., 2003; Liddament et al., 2004; Wiegand et al., 2004; Zheng et al., 2004)] as opposed to APOBEC3G [(5′ CpC preference, or GpG on plus strand (Harris et al., 2003; Lecossier et al., 2003; Suspene et al., 2004)] was being expressed in a subset of cells. Presumably 3D-PCR was
  • Poliovirus VP1 PCR products from ten patients with post-vaccinal acute flaccid paralysis were examined. A smaller 480 bp nested segment was targeted and the denaturation conditions were investigated by using the primer pair UG1/UC1 (Guillot et al., 2000). Calibration using cloned DNA showed that the reference Sabin 1 sequence was amplified by using denaturation temperatures from 95 to 91° C., but not from 90 to 80° C. Sabin 2 and 3 targets were subtly different from Sabin 1 in that they could not be amplified below 92° C.
  • the length of the window affects the ability to discriminate between alleles differing in GC content. The longer the DNA segment the poorer the discrimination. The inverse is true to the point that an attempt was made to identify a single point mutation in a small window of as little as 30 bases.
  • the case chosen was the p21 ras gene and the “famous” mutation in codon 12 that transforms the gene into an oncogene.
  • the sequences of the two alleles are shown in FIG. 4A .
  • the PCR primers are underlined as is the single G residue in the wild type sequence that is mutated to T in the oncogene.
  • the “window” between the two primers is 29bp.
  • the methods of the invention namely, 3D-PCR can be used to differentially amplify AT-enriched genomes compared with the parental genome.
  • retroviral hypermutants are preferred targets for 3D-PCR, it can be applied to any sample in which there is a mutant or mutant spectrum.
  • 3D-PCR allows differential amplification of genomes that differ by just a few GC ⁇ AT transitions.
  • the degree of substitution directly affects the melting temperature of the DNA, the lower the denaturation temperature, the more substituted the genomes that are amplified.
  • the conditions can be optimized for each segment.
  • the method allows differential amplification, it is not quantitative per se. However, coupled to limiting dilution of input DNA, it is possible to quantify the fraction of AT-rich genomes within a sample.
  • 3D-PCR can be used to amplify AT-rich bacterial 16S rDNA sequences within a heterogeneous natural sample, neo-deaminated immunoglobulin V regions, or promoter regions that have undergone extensive 5-MeC deamination following extensive methylation.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US11/321,048 2005-12-30 2005-12-30 Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids Abandoned US20070154892A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US11/321,048 US20070154892A1 (en) 2005-12-30 2005-12-30 Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids
JP2008548048A JP2009524412A (ja) 2005-12-30 2006-12-28 核酸の混合物におけるｐｃｒによる突然変異核酸の示差的増幅
CA002635109A CA2635109A1 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids
PCT/IB2006/004187 WO2007091125A2 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids
US12/086,897 US8541206B2 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids
EP06849524.1A EP1979492B1 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids
US12/691,377 US20100184017A1 (en) 2005-12-30 2010-01-21 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US11/321,048 US20070154892A1 (en) 2005-12-30 2005-12-30 Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/086,897 Continuation US8541206B2 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids
US12/691,377 Continuation US20100184017A1 (en) 2005-12-30 2010-01-21 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids

Publications (1)

Publication Number Publication Date
US20070154892A1 true US20070154892A1 (en) 2007-07-05

Family

ID=38224885

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/321,048 Abandoned US20070154892A1 (en) 2005-12-30 2005-12-30 Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids
US12/086,897 Expired - Fee Related US8541206B2 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids
US12/691,377 Abandoned US20100184017A1 (en) 2005-12-30 2010-01-21 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/086,897 Expired - Fee Related US8541206B2 (en) 2005-12-30 2006-12-28 Differential amplification of mutant nucleic acids by PCR in a mixture of nucleic acids
US12/691,377 Abandoned US20100184017A1 (en) 2005-12-30 2010-01-21 Differential amplification of mutant nucleic acids by pcr in a mixture of nucleic acids

Country Status (5)

Country Link
US (3) US20070154892A1 (ja)
EP (1) EP1979492B1 (ja)
JP (1) JP2009524412A (ja)
CA (1) CA2635109A1 (ja)
WO (1) WO2007091125A2 (ja)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2183379A2 (en) * 2007-08-01 2010-05-12 Dana Farber Cancer Institute Enrichment of a target sequence
EP2314680A1 (en) * 2008-07-02 2011-04-27 ARKRAY, Inc. Method for amplification of target nucleic acid sequence, method for detection of mutation by using the method, and reagents for use in the methods
US9133490B2 (en) 2012-05-16 2015-09-15 Transgenomic, Inc. Step-up method for COLD-PCR enrichment
CN104946626A (zh) * 2011-01-12 2015-09-30 积水医疗株式会社 离子交换色谱法用洗脱液以及核酸链的分析方法
US9284603B2 (en) 2010-01-21 2016-03-15 Arkray, Inc. Target sequence amplification method, polymorphism detection method, and reagents for use in the methods
US9447460B2 (en) 2011-01-12 2016-09-20 Sekisui Medical Co., Ltd. Method for detecting single nucleotide polymorphisms
US9957556B2 (en) 2010-03-08 2018-05-01 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
US10913977B2 (en) 2013-07-24 2021-02-09 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable enrichment of minor DNA alleles by limiting denaturation time in PCR or simply enable enrichment of minor DNA alleles by limiting the denaturation time in PCR
US11008606B2 (en) * 2014-10-10 2021-05-18 Cold Spring Harbor Laboratory Random nucleotide mutation for nucleotide template counting and assembly
US11130992B2 (en) 2011-03-31 2021-09-28 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable multiplex COLD-PCR
US11174511B2 (en) 2017-07-24 2021-11-16 Dana-Farber Cancer Institute, Inc. Methods and compositions for selecting and amplifying DNA targets in a single reaction mixture
US11371090B2 (en) 2016-12-12 2022-06-28 Dana-Farber Cancer Institute, Inc. Compositions and methods for molecular barcoding of DNA molecules prior to mutation enrichment and/or mutation detection

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8288094B2 (en) 2008-04-09 2012-10-16 Institut Pasteur APOBEC3 mediated DNA editing
WO2014018093A1 (en) * 2012-07-26 2014-01-30 Illumina, Inc. Compositions and methods for the amplification of nucleic acids
US9279146B2 (en) 2012-12-21 2016-03-08 Roche Molecular Systems, Inc. Compounds and methods for the enrichment of mutated nucleic acid from a mixture
US9873908B2 (en) 2013-11-27 2018-01-23 Roche Molecular Systems, Inc. Methods for the enrichment of mutated nucleic acid from a mixture

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5461143A (en) * 1991-03-18 1995-10-24 The Scripps Research Institute Oligosaccharide enzyme substrates and inhibitors: methods and compositions
US7179589B2 (en) * 1998-10-08 2007-02-20 Dynametrix Ltd. Detection of nucleic acid polymorphism

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5512462A (en) * 1994-02-25 1996-04-30 Hoffmann-La Roche Inc. Methods and reagents for the polymerase chain reaction amplification of long DNA sequences
US8283148B2 (en) * 2002-10-25 2012-10-09 Agilent Technologies, Inc. DNA polymerase compositions for quantitative PCR and methods thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5461143A (en) * 1991-03-18 1995-10-24 The Scripps Research Institute Oligosaccharide enzyme substrates and inhibitors: methods and compositions
US7179589B2 (en) * 1998-10-08 2007-02-20 Dynametrix Ltd. Detection of nucleic acid polymorphism

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100203532A1 (en) * 2007-08-01 2010-08-12 Dana-Farber Cancer Institute Enrichment of a Target Sequence
EP2183379A4 (en) * 2007-08-01 2011-08-10 Dana Farber Cancer Inst Inc ENRICHMENT OF A TARGET SEQUENCE
US8455190B2 (en) 2007-08-01 2013-06-04 Dana-Farber Cancer Institute, Inc. Enrichment of a target sequence
CN101815789B (zh) * 2007-08-01 2013-06-12 达纳-法伯癌症研究院有限公司 靶序列的富集
AU2008282780B2 (en) * 2007-08-01 2014-04-17 Dana- Farber Cancer Institute Enrichment of a target sequence
EP2183379A2 (en) * 2007-08-01 2010-05-12 Dana Farber Cancer Institute Enrichment of a target sequence
EP2314680A1 (en) * 2008-07-02 2011-04-27 ARKRAY, Inc. Method for amplification of target nucleic acid sequence, method for detection of mutation by using the method, and reagents for use in the methods
EP2314680A4 (en) * 2008-07-02 2011-12-14 Arkray Inc METHOD FOR THE AMPLIFICATION OF A TARGET NUCLEIC ACID SEQUENCE, METHOD FOR METHODIC DETECTION USING THE METHOD AND REAGENT FOR USE IN THE PROCEDURES
US9115391B2 (en) 2008-07-02 2015-08-25 Arkray, Inc. Method of detecting a polymorphism at a polymorphism site
US9284603B2 (en) 2010-01-21 2016-03-15 Arkray, Inc. Target sequence amplification method, polymorphism detection method, and reagents for use in the methods
US9957556B2 (en) 2010-03-08 2018-05-01 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
US11174510B2 (en) 2010-03-08 2021-11-16 Dana-Farber Cancer Institute, Inc. Full COLD-PCR enrichment with reference blocking sequence
CN104946626A (zh) * 2011-01-12 2015-09-30 积水医疗株式会社 离子交换色谱法用洗脱液以及核酸链的分析方法
US9481881B2 (en) 2011-01-12 2016-11-01 Sekisui Medical Co., Ltd. Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains
US9447460B2 (en) 2011-01-12 2016-09-20 Sekisui Medical Co., Ltd. Method for detecting single nucleotide polymorphisms
US11130992B2 (en) 2011-03-31 2021-09-28 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable multiplex COLD-PCR
US9133490B2 (en) 2012-05-16 2015-09-15 Transgenomic, Inc. Step-up method for COLD-PCR enrichment
US10913977B2 (en) 2013-07-24 2021-02-09 Dana-Farber Cancer Institute, Inc. Methods and compositions to enable enrichment of minor DNA alleles by limiting denaturation time in PCR or simply enable enrichment of minor DNA alleles by limiting the denaturation time in PCR
US11008606B2 (en) * 2014-10-10 2021-05-18 Cold Spring Harbor Laboratory Random nucleotide mutation for nucleotide template counting and assembly
US11371090B2 (en) 2016-12-12 2022-06-28 Dana-Farber Cancer Institute, Inc. Compositions and methods for molecular barcoding of DNA molecules prior to mutation enrichment and/or mutation detection
US11174511B2 (en) 2017-07-24 2021-11-16 Dana-Farber Cancer Institute, Inc. Methods and compositions for selecting and amplifying DNA targets in a single reaction mixture

Also Published As

Publication number Publication date
CA2635109A1 (en) 2007-08-16
EP1979492A2 (en) 2008-10-15
WO2007091125A2 (en) 2007-08-16
US8541206B2 (en) 2013-09-24
JP2009524412A (ja) 2009-07-02
US20100184017A1 (en) 2010-07-22
EP1979492B1 (en) 2014-10-29
WO2007091125A3 (en) 2009-02-12
US20110003282A1 (en) 2011-01-06

Similar Documents

Publication Publication Date Title
US20070154892A1 (en) Differential amplification of mutant nucleic acids by PCR in a mixure of nucleic acids
Suspene et al. Recovery of APOBEC3-edited human immunodeficiency virus G→ A hypermutants by differential DNA denaturation PCR
Zhao et al. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron
CN111394430B (zh) 一种基于CRISPR-Cas12a耦合增强型链置换扩增的检测系统及其应用
US20230392191A1 (en) Selective degradation of wild-type dna and enrichment of mutant alleles using nuclease
Yan et al. HIV DNA is heavily uracilated, which protects it from autointegration
JP5805064B2 (ja) 対立遺伝子変種を検出するための方法、組成物、およびキット
Yu et al. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome
JP3561523B2 (ja) 核酸分子を特性化する方法
EP2488656B1 (en) Multiple displacement amplification
EP2623613B1 (en) Increasing confidence of allele calls with molecular counting
Zanini et al. Error rates, PCR recombination, and sampling depth in HIV-1 whole genome deep sequencing
JP2005509440A (ja) 多重オリゴヌクレオチド付加および標的増幅
US20100068712A1 (en) Oligonucleotides for use in allele-specific pcr
Diaz et al. Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase
Suspène et al. Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics
US6878530B2 (en) Methods for detecting polymorphisms in nucleic acids
WO2016019455A1 (en) Site-specific endonuclease guided rolling circle amplification
WO2006070666A1 (ja) 遺伝子多型の同時検出方法
US20120322665A1 (en) System and method for detection of hiv-1 clades and recombinants of the reverse transcriptase and protease regions
JP2007295838A (ja) 核酸増幅を利用した核酸ミスマッチ検出方法
US20060051749A1 (en) Polymorphism and haplotype scoring by differential amplification of polymorphisms
KR20220097234A (ko) 중합효소연쇄반응 관련 교차 오염물 제거를 위한 프라이머 세트
US7402382B2 (en) GEDAP method (genotyping based on diagnostic amplification products) for detecting and/or preventing genotyping errors from amplification products of a polymorphic focus
JP2008136436A (ja) １本鎖ｄｎａ結合蛋白質を用いた核酸の変異検出方法

Legal Events

Date Code Title Description
AS Assignment

Owner name: INSTITUT PASTEUR, FRANCE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WAIN-HOBSON, SIMON;VARTANIAN, JEAN-PIERRE;REEL/FRAME:017654/0310

Effective date: 20060110

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION