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WO2008064687A1 - Procédé d'amplification spécifique d'allèle à fidélité accrue - Google Patents

Procédé d'amplification spécifique d'allèle à fidélité accrue Download PDF

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Publication number
WO2008064687A1
WO2008064687A1 PCT/DK2007/050178 DK2007050178W WO2008064687A1 WO 2008064687 A1 WO2008064687 A1 WO 2008064687A1 DK 2007050178 W DK2007050178 W DK 2007050178W WO 2008064687 A1 WO2008064687 A1 WO 2008064687A1
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Prior art keywords
primer
nucleic acid
amplification
pcr
stop
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PCT/DK2007/050178
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English (en)
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Lars Christian Von Gersdorff
Tomas Ussing
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Fluimedix
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Publication of WO2008064687A1 publication Critical patent/WO2008064687A1/fr

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the invention relates to a method with which an increased fidelity in the product of an Allele Specific Amplification (ASA) -assay, can be accomplished.
  • the method is based on "primer competition" and will aid in minimising the amount of non-specific amplification product that may occur in a normal ASA amplification, when the interrogated template does not contain the target sequence.
  • ASA Allele Specific Amplification
  • genotyping assays involves a DNA amplification method known as the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • oligonucelotides two short DNA sequences initiating amplification start and stop of the desired DNA sequence.
  • the two oligonucleotides are referred to as a primer set comprising of a forward and a reverse primer.
  • the design of the primers decides which fragment of a given DNA template is amplified.
  • a method commonly used for SNP and INDEL detection is amplification refractory mutation system (ARMS) also known as allele specific amplification (ASA) 1 ".
  • ARMS amplification refractory mutation system
  • ASA allele specific amplification
  • WT wild type
  • MT mutant perfect match
  • the primers are commonly designed with the mismatch near the 3'-end of the oligonucletide sequence, as amplification in a PCR is most sensitive to mismatches in that part of the primer. It is imperative that the DNA polymerase used for the amplification lacks 3' to 5' exonuclease activity, as this ability enables the polymerase to modify the sequence of the mismatching primer.
  • a primer with a GC clamp (3 or more GC bases at the 3'-end of the primer) has a high 3'-end stability and thus increases the risk of unspecific amplification. It has been suggested that introducing an extra mismatch near the 3'-end of the primer further increases the sensitivity of the primer 1 " leading to higher fidelity. However this also affects primer efficiency, and may dramatically decrease amplification yield.
  • ASA IV Other variants of the ASA method have been developed such as bi-directional ASA IV .
  • two set of primers with overlapping target sequence are designed in a way so one forward primer matches the WT allele and one reverse primer matches the MT allele.
  • the remaining two primers are designed so the two potential amplification fragments are of significantly different size.
  • either a short or a long or both fragments will be amplified allowing detection of homozygote and heterozygote genotypes.
  • subsequent fragment size analysis such as melt curve analysis or gel electrophoresis, mandatory.
  • V stop oligonucleotides are designed to bind within the amplicon of two normal oligonucleotide PCR primers.
  • the normal oligonucleotide PCR primers are extended until they reach the stop oligonucleotide and by including a ligase in the reaction the extended normal oligonucleotide PCR primer is ligated to the stop primer hence producing a blocking primer which screens the template during the subsequent cycles.
  • the normal primer must anneal to the template
  • the stop primer must anneal to the same template strand
  • the normal primer must be extended and the extended normal primer must be ligated to the stop primer.
  • only one incident must occur for blocking of the unspecific template nucleic acid as only the stop oligonucleotide must anneal to the template before blocking of the unspecific template is obtained.
  • the TSI-PCR setup is also dependent on an extra enzymatic step, namely ligation of the extended normal primer to the stop primer, leading to a longer total assay time.
  • TSI-PCR setup Another limitation of the TSI-PCR setup is that the polymerase used in the setup must lack 5' to 3' exonucleoase activity or the stop oligonucleotide will be digested by the polymerase. Furthermore the TSI-PCR setup is designed to block larger parts of unspecific nucleic acid template and not for SNP detection.
  • the ASA PCR method is combined with the use of peptide nucleic acid (PNA) clamping primers, also called PNA clamping".
  • PNA peptide nucleic acid
  • the principle of the PNA clamping method is to introduce a PNA blocking primer that matches the allelic variant of the target sequence along with an extra annealing step in the thermal cycling profile to the ASA PCR setup.
  • the PNA primer has a significantly higher annealing temperature to the target nucleic acid (typically DNA or RNA) sequence the introduction of the extra annealing step at a higher temperature than the ordinary annealing step will result in the PNA clamping primer to bind to the allelic variant (if present) and this way blocking the binding site of the ordinary primer and preventing unspecific binding and amplification.
  • the thermal cycling period will be extended and a longer assay time is needed.
  • the PNA clamping primer may bind and block the true priming site for the ordinary primer and thus leading to a low yield.
  • the PNA probes may also displace specific bound DNA primers in the second annealing step again leading to low yield.
  • an allele specific primer competition (ASPC) method was developed.
  • This method comprises the use of at least one common primer and at least two discriminating opposite directed primers.
  • One of the discriminating primers is modified in the 3'-end in a way that prevents primer extension, in particular enzymatic primer extension, this primer is termed the STOP primer.
  • the other discriminating primer allows for primer extension, in particular enzymatic primer extension, this primer is termed the normal primer.
  • the two discriminating primers designed to overlap the flanking sequence around the variant part of the target sequence.
  • the two discriminating primers are designed as follows: one perfectly matching the first variant of the sequence, sequence 1, and one perfectly matching the second variant of the sequence, e.g. the variant comprising a mutation as compared to sequence 1, sequence 2. If the first primer is a normal primer, then the second primer is a STOP primer and vice versa. In a reaction where only the first variant of the target sequence is present (sequence 1), and the first primer is a normal primer, amplification will occur as the discriminating primer that predominantly will anneal to the target sequence is the primer perfectly matching the first variant of the target sequence.
  • the method as described above is not limited to the use of primers perfectly matching the nucleic acid sequence to be interrogated (e.g. sequence 1 and sequence 2 above), it is sufficient that one of the two discriminating primers has a higher affinity, as compared to the other discriminating primer, for one of the sequences. Accordingly, in a preferred embodiment either the normal primer or the STOP primer has a higher affinity to a nucleic acid sequence which differs by at least one base pair from the nucleic acid sequence to be interrogated, as it has to the nucleic acid sequence to be interrogated, whereas the other discriminating primer has a higher affinity to the nucleic acid sequence to be interrogated.
  • more than one nucleic acid sequence to be interrogated such as at least 2, or at least 3, or at least 4, or at least 6, or at least 10, or at least 15, or at least 20, is present and the method of the invention is executed as a one-step method. Accordingly, in a first aspect the present invention relates to:
  • a method of analysing a nucleic acid sequence by nucleic acid amplification comprising the steps of a) defining at least one nucleic acid sequence to be interrogated b) providing the at least one nucleic acid sequence to be interrogated c) providing at least one common primer d) providing at least two discriminating primers, wherein a. at least one of the discriminating primers is modified in the 3'-end in a way that prevents extension (STOP primer), b. at least one of the discriminating primers allows for extension (normal primer), and c. the at least one STOP primer and the at least one normal primer is opposite directed to the at least one common-primer d.
  • either the at least one normal primer or the at least one STOP primer has a higher affinity to a nucleic acid sequence which differs by at least one base pair from the nucleic acid sequence to be interrogated, as it has to the nucleic acid sequence to be interrogated, whereas the at least one other discriminating primer has a higher affinity to the nucleic acid sequence to be interrogated.
  • nucleic acid sequence is intended to encompass ribonucleic acids (such as RNA, imRNA, piRNA, tRNA, rRNA, ncRNA, gRNA, shRNA, siRNA, snRNA, miRNA, snoRNA), and deoxyribonucleic acids (such as DNA, mtDNA, cDNA, artificial chromosomes, e.g. bacterial artificial chromosome, yeast artificial chromosome and human artificial chromosome) as well as analogues of nucleic acids (such as GNA, PNA, TNA and LNA).
  • ribonucleic acids such as RNA, imRNA, piRNA, tRNA, rRNA, ncRNA, gRNA, shRNA, siRNA, snRNA, miRNA, snoRNA
  • deoxyribonucleic acids such as DNA, mtDNA, cDNA, artificial chromosomes, e.g. bacterial artificial chromosome, yeast artificial chromosome
  • the STOP primer may be modified in the 3'-end by addition of biotin, phosphate, di- deoxynucleotide triphosphates ("ddNTPs"), also referred to as chain terminating ddNTPs, and ethylene glycol linkers on the 3'OH.
  • ddNTPs di- deoxynucleotide triphosphates
  • ethylene glycol linkers on the 3'OH.
  • the 3'end of the nucleic acid may be capped using non-standard bases such as, but not limited to, AEGIS bases.
  • non-standard bases such as, but not limited to, AEGIS bases.
  • amplification systems for example but not limited to PCR, TMA, SDA, NASBA, depend in part upon the ability of a nucleic acid polymerase to extend off the 3'hydroxyl of an oligonucleotide primer.
  • the primers modified in the 3'-end in a way that prevents enzymatic primer extension are designed to match a different sequence around the nucleic acid sequence to be interrogated, e.g. the SNP or INDEL object of the investigation, than the competing extendable normal primer. This allows for design of primers with a more homogeneous annealing temperature, thus providing a more robust assay.
  • more than one STOP primer is incorporated, so that the presence of additional mutations will be taken into account (i.e. in a SNP detecting assay STOP primers with a perfect match with allelic variant 2, allelic variant 3, allelic variant 4 etc. - genotype, may be designed and included in an assay devised to amplify and detect only wild-type - allelic variant 1)
  • the method may be applied to a Multi-plex PCR assay, where one, more or all of the involved assays may be designed according to the described method of the present invention.
  • one or more nucleic acids are added to the 5'-end of the at least one normal primer, and at least one of the added nucleic acids does not match the sequence to be interrogated. In one preferred embodiment the addition of the one or more nucleic acids does not result in the formation of a hairpin loop.
  • the oligonucleotide tag part of the normal extendable primer is deliberately designed to form a hairpin loop which e.g. can be used as a signaling probe like Scorpion primers v ⁇ " or Molecular Beacons" * .
  • nucleic acids such as less than 20 nucleic acids, such as less than 15 nucleic acids or less than 10 nucleic acids, preferably less that 8 nucleic acids, such as less than 5 nucleic acids, such as 2 or 3 or 4 nucleic acids, are added to the at least one normal primer.
  • the STOP primers are used to block false priming sites in regions of the template with high sequence similarity to the nucleic acid sequence to be interrogated.
  • a STOP primer with a higher affinity for the false priming site than normal extendable primer but with a lower affinity for the true priming site than the normal primer, the false priming site will be blocked by the STOP primer by competition and amplification of an unwanted part of the template is avoided.
  • An example of such a situation could be if a SNP is located in a gene which has a pseudogene and the SNP is desired analyzed by ASA PCR.
  • a pseudogene is a non-functioning relative of a known gene that has lost its protein-coding ability or is no longer expressed in the cell x .
  • Pseudogenes often share a very high sequence similarity to it origin gene thus providing many false priming sites when designing ordinary ASA PCR primers. Genotyping SNPs in a gene which has pseudogenes often require separation of the origin gene from the pseudogene in the sample before genotyping using ASA PCR analysis. But by blocking false priming sites in the pseudogenes using STOP primers the separation step can be eliminated.
  • the difference in annealing temperature between the at least one STOP primer and the at least one normal primer is less than 5 0 C, such as less than 4 0 C, such as less than 3 0 C or less than 2 0 C, preferably less that I 0 C or less than 0.5 0 C or less than 0.1 0 C
  • the method of the present invention may be applied for genotyping of the nucleic acid sequence or sequences to be interrogated.
  • the nucleic acid sequence or sequences to be interrogated may comprise two or more alleles; in particular it may of interest to apply the method of the present invention to sequences comprising Single Nucleic Polymorphisms. In a further aspect it may be of interest to use the method of the present invention for detection of homozygote and heterozygote forms of allelic variants.
  • the present invention relates to a device for analysis of a nucleic acid sequence by nucleic acid amplification according the method of the present invention.
  • a device for analysis of a nucleic acid sequence by nucleic acid amplification according the method of the present invention.
  • the device may comprise a reading device, e.g. a photometer, and may be portable.
  • the normal extendable primer is modified in 5'-end with a short oligonucleotide (a tag) which is non-complementary to the target nucleic acid sequence to be interrogated.
  • the stop primer is not modified with the oligonucleotide tag.
  • a short oligonucleotide tag By adding a short oligonucleotide tag to the normal extendable primers may the same effect as a touch down PCR (TD PCR) X1 setup provides is achieved without gradually lowering but maintaining a constant annealing temperature.
  • TD PCR touch down PCR
  • X1 setup By using the tag modified normal extendable primer setup it is also possible to increase the annealing temperature during the cycles in a normal PCR thermal cycling profile.
  • the oligonucleotide tags should be designed individually for the discriminating primer variants, in order to obtain primers with homogenous annealing temperature of the two primer variants.
  • the invention is particularly relevant when performing PCR in systems (e.g. portable microsystems) where one constant exact temperature is easier to obtain rather than precise control of temperature in a given range.
  • WT sequence 1st cycle, provided sample nucleic acid is primary template: ...5 ' -attgattatttcccgggaacccataacaaattacttaaaaacc-3 ' ... WT sequence
  • the WT allelic variant is present as nucleic acid template (in this example DNA template)
  • nucleic acid template in this example DNA template
  • perfect annealing of the WT tag reverse primer occurs and no annealing of the MT reverse stop primer occurs hence the polymerase can extend the WT primer.
  • the underlined part of the tag WT reverse primer is the oligonucleotide tag.
  • the inclusion of the tag sequence in the amplified DNA fragment provides annealing of the entire primer including the tag (the underlined part of the sequences), thus increasing optimal annealing temperature and hence increasing yield.
  • FIG 1 The principle of allele specific primer amplification with fidelity enhancing means (ASPC, the method of the present invention).
  • Step 1 Primer annealing.
  • SEQl forward normal primer In the presence of a SEQl allele the SEQl forward normal primer will predominantly anneal to the DNA template, and amplification will occur in step 2.
  • SEQ2 forward STOP primer In the presence of the SEQ2 allele the SEQ2 forward STOP primer will predominantly anneal in step 1 and subsequently prevent amplification in step 2.
  • SEQ2 forward normal primer and SEQl forward STOP primer allows for a positive control for detection of the SEQ2 allele. Combination of the two parallel reactions provides an assay that can detect heterozygote and both types of homozygote genotypes.
  • the normal primer and STOP primer can equally well be reverse primers.
  • Figure 2 MicroGel electrophoresis: Lane description (from left to right) : DNA reference ladder; PCR no 6; PCR no 5; PCR no 4; PCR no 3; PCR no 2; PCR no 1; DNA reference ladder.
  • the DNA reference ladder used was TrackltTM 50 bp DNA ladder (Invitrogen, Ca, USA) with DNA markers in 50 bp increments. Approximately 1/3 from the top of the lanes loaded with ladder, a band brighter than the other DNA ladder bands is observed. This is the 350 bp DNA ladder band. In PCRs no 1 - 6 amplification of a fragment in the size range of 100 - 150 bp is observed, which correlates with the 135/136 bp fragment which was expected amplified.
  • PCRs no 1 and 2 are 10 ⁇ l reactions of the MT mix
  • PCRs no 3 and 4 are 10 ⁇ l reactions of the WT mix
  • PCRs no 5 and 6 are 10 ⁇ l reactions of the WT+MT- STOP mix.
  • Best amplification is observed in PCRs no 1 and 2, which also is expected as the DNA template is a homozygote MT CYP2C19*2 carrier.
  • High amplification is also observed on PCRs no 3 and 4, which is not expected as these PCRs were reactions from the WT primer mix.
  • PCRs no 5 and 6 significantly lower amplification than in PCRs no 3 and 4 is observed.
  • PCRs no 5 and 6 were reactions from the WT+MT-STOP primer mix.
  • Figure 3 - CYP2D6 gene has two pseudo genes located directly upstream the CYP2D6 gene.
  • Figure 4 Image of the MicroGel from the PCRs in Example 4; Lane description (from left to right) : DNA reference ladder; PCR no 10; PCR no 9; PCR no 8; PCR no 7; PCR no 6; PCR no 5; PCR no 4; PCR no 3; PCR no 2; PCR no 1; DNA reference ladder.
  • the DNA reference ladder used was TrackltTM 50 bp DNA ladder (Invitrogen, Ca, USA) with DNA markers in 50 bp increments. Amplification is observed in PCRs no 1, 3, 6, 7 and 8.
  • the amplified fragment is in the size range of 100 - 150 bp (between step no 2 and 3 counted from the bottom) which correlates with the 135/136 bp fragment which was expected amplified.
  • the assay is designed so amplification should only occur in PCRs no 1, 3, 6 and 8 as the WT mix and WT-S mix only should provide amplification when a homozygote WT sample template was present (PCRs 1 and 3), and the MT mix and MT-S mix only should provide amplification when a homozygote MT sample template was present (PCRs 6 and 8). However amplification is also observed in PCR no 7, which is the MT-S mix PCR with homozygote WT sample template.
  • Figure 5 Amplification curves from the ASA PCR based assay from experimental example 5. From the ordinary ASA PCR amplification curves a high level of amplification is observed in both the WT-S and the MT-S PCRs, highest in the MT-S PCR. A high level of amplification should only have occurred in the WT-S PCR while no or very low amplification should have occurred in the MT-S PCR, as the template DNA only contained the WT allelic variant.
  • FIG. 6 Amplification curves from the ASPC PCR based assay from experimental example 5.
  • the ASPC PCR amplification curves show a high level of amplification in the WT PCR and a low level (approximately equal to the level of the negative controls) of amplification in the MT PCR.
  • the amplification yield in the WT PCR is a approximately factor 5 higher than the MT PCR amplification yield.
  • ASA PCR setup a high level of amplification should only have occurred in the WT PCR while no or very low amplification should have occurred in the MT PCR, as the template DNA only contained the WT allelic variant.
  • Figure 7 Image of the MicroGel from the PCRs in experimental example 6.
  • Lane description (from left to right) : DNA reference ladder; PCR no 8; PCR no 7; PCR no 6; PCR no 5; PCR no 4; PCR no 3; PCR no 2; PCR no 1; DNA reference ladder.
  • the DNA reference ladder used was TrackltTM 50 bp DNA ladder (Invitrogen, Ca, USA) with DNA markers in 50 bp increments.
  • the ASPC PCR based assay is designed so amplification should only occur in PCRs no 1, 3, 6 and 8 as the WT mix only should provide amplification when a homozygote WT sample template or heterozygote sample template was present (PCRs 1 and 3), and the MT mix only should provide amplification when a homozygote MT sample template or a heterozygote sample template was present (PCRs 6 and 4). Amplification is observed in PCRs no 1, 3, 4 and 6. By counting the steps in DNA reference ladder lanes it is observed that the amplified fragment is in the size range of 100 - 150 bp (between step no 2 and 3 counted from the bottom) which correlates with the 135/136 bp fragment which was expected amplified.
  • Two sets of primers were designed to discriminate between the wild type allele of the CYP2C19 gene variant regarded CYP2C19*1 and the mutant allele variant CYP2C19*2.
  • Normal ASA is difficult as the flanking regions of the CYP2C19*2 SNP are very GC-rich (CYP2C19*2 sequence: 5'...TCCCGGGAA...3', the underlining indicates the SNP position) meaning that a single bp mismatch in this region still provides a strong binding primer.
  • the SNP is a G to A shift, meaning that detection of the MT allele without having unspecific amplification from the in WT reaction is difficult.
  • the discriminating primer is the reverse primer, where two versions (WT and MT) were designed to amplify a 135 respectively 136 bp fragment of the CYP2C19 gene. An extra bp is added to the 5'-end of the MT primer to homogenise the melting temperature (Tm) of the WT and MT primers.
  • Tm melting temperature
  • the STOP primers were modified with a phosphate group in the 3'-end to prevent extension of the primer during the amplification. Both primers share the same forward primer. See sequences below.
  • Reverse primer MT 5' - 3' (SEQ ID NO: 3) : TTTAAGTAATTTGTTATGGGTTCCT
  • Non-polymerase extendable STOP primers are non-polymerase extendable STOP primers:
  • the MT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and 2C19*2 PI RMTn+T primer.
  • the WT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and 2C19*2 PI RWTn primer.
  • the WT+MT-STOP mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 PI RWTn primer and 2C19*2 PI RMTn+T 3'P.
  • All primers were HPLC purified and delivered by TAG Copenhagen A/S (Denmark). In all three mixes a final concentration of 0.1 unit Taq DNA polymerase (Ampliqon, Denmark), 0.2 mM of each dNTPs: dATP, dTTP, dGTP and dCTP (Ampliqon, Denmark), Ix Ammonia Reaction Buffer (tween free) (Ampliqon, Denmark), 2.5 mM MgCI 2 and 12.5 ng human genomic DNA. The DNA template used in all three mixes was human genomic DNA previously genotyped as homozygote MT regarding the CYP2C19*2 allele.
  • PCRs were tested by gel electrophoresis using an in-house fabricated MicroGel electrophoresis setup.
  • a 2.5% agarose gel prepared in 0.5 X TBE buffer and stained with ethidium bromide (1.5 ⁇ l ethidum bromide for 10 ml agarose gel solution) were cast with the dimensions of 30x54x3 mm (width x length x thickness).
  • the gel were covered in 1 X TBE buffer and loaded with 1 ⁇ l sample or DNA reference ladder pr. lane, and run at 35 V for 45 minutes. After 45 min. the gel was exposed with UV-light and an image of the gel was taken, see fig. 2. On fig. 2 it can be observed that amplification occurs in all three PCR mixes.
  • PCR no 1 and 2 which is also expected as this mix included the MT allele perfect match reverse primer.
  • amplification is also observed in PCRs no 3 and 4, is not expected as this mix included WT allele perfect match reverse primer. This indicates the difficulties there is with ordinary ASA discrimination between SNPs in GC-rich regions.
  • PCRs no 5 and 6 a significantly lower amplification is observed compared to PCRs no 3 and 4.
  • PCRs no 5 and 6 included the WT allele perfect match reverse primer and the MT allele perfect match reverse STOP primer.
  • two sets of reverse primers are designed to discriminate between the wild type allele (WT) of the CYP2D6 gene and the CYP2D6*4 SNP allele variant (MT).
  • the SNP is located in a GC-rich region of the CYP2D6-gene (CYP2D6*4 sequence: 5'-...CCCCCAGGACGCCCC..-3' (SEQ ID NO: 6), the underlined bp marks the CYP2D6*4 SNP position) which would make normal ASA PCR difficult, as a single bp shift in this region will not provide enough shift in the optimal annealing temperature for the normal ASA primers to provide sufficient discrimination between the WT allele and the MT allele.
  • a primer designed to overlap the SNP will have a 3'-end with a high GC-content, providing a very strong binding primer. Both conditions increases the risk of unspecific binding and hence unspecific amplification, which is problematic in an end- point readout assay based on amplification in the presence of a specific sequence and no amplification in the presence of a one bp variant of the same sequence.
  • a stop primer which is non-extendable by a polymerase
  • the stop primer will bind over the normal primer and by competition block unspecific binding of the normal primer and hence reducing unspecific amplification. So STOP versions of the two CYP2D6*4 discriminating reverse primers are also designed by modifying the 3'-end of the primers so extension by a polymerase is not possible. The reverse primers share the same forward primer. See sequences below:
  • Non-polymerase extendable primers are non-polymerase extendable primers:
  • Amplified sequence MT, 173 bp (SEQ ID NO: 11) :
  • the WT PCR setup comprises of the 2D6*4 F primer, the 2D6*4 R WT primer and the 2D6*4 R MT STOP primer.
  • the 2D6*4 R WT primer will predominantly bind and amplification will occur.
  • the MT PCR setup comprises of the 2D6*4 F primer, the 2D6*4 R MT primer and the 2D6*4 R WT STOP primer. This setup is the opposite of the WT reaction so in the presence of a MT allele in the template NA, the 2D6*4 R MT primer will predominantly bind and amplification will occur. If only WT alleles are present in the template nucleic acid the
  • homozygote carriers of the WT and the MT allele as well as heterozygote carriers can be identified. If the nucleic acid sample is from a homozygote WT carrier, amplification will only occur in the WT PCR and not in the MT PCR. If the nucleic acid sample is from a homozygote MT carrier, amplification will not occur in the WT PCR but only in the MT PCR. Heterozygote carriers are identified by amplification in both the WT PCR and MT PCR.
  • two sets of forward primers are designed to discriminate between the wild type allele (WT) of the CYP2D6 gene and the CYP2D6*7 SNP allele variant (MT).
  • WT wild type allele
  • MT CYP2D6*7 SNP allele variant
  • Designing PCR primers for the CYP2D6 gene can be very difficult as the CYP2D6 gene has two pseudo genes located directly upstream the CYP2D6 gene, see figure 3.
  • the two pseudo genes CYP2D8P and CYP2D7 share 89% and 97% respectively of the CYP2D6 sequenceTM thus the pseudo genes provide many false priming sites genes for PCR primers designed specifically for amplification of a fragment in the CYP2D6 gene.
  • the forward primers are designed to overlap the CYP2D6*7 SNP (CYP2D6*7 sequence: 5'-...GATCCTACATCCGGATG...-3', the underlined bp marks the CYP2D6 *7 SNP position, the SNP is a A to C change)
  • the forward WT primer has a 100% match in the CYP2D7 gene and a 95% match in the CYP2D8P gene, see sequences below: 5'-...TGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGATGTGCAGCGT...-3' CYP2D6 (SEQ ID NO: 14)
  • the underlined bps indicate differences in the pseudo genes compared to the CYP2D6 gene and the double underlined bp indicates the position of the CYP2D6*7 SNP in the CYP2D6 gene.
  • ASA PCR setup should be used to identify the CYP2D6*7 variant, the CYP2D6 gene would have to be isolated before the ASA PCR and hence sample preparation will require extra steps. If ASA PCR setup is run without isolating the CYP2D6 gene from the pseudo genes there is a high risk of false positive reactions.
  • stop primers that blocks the binding sites for the normal primer in the pseudo genes, unspecific amplification of the pseudo genes and hence the false positive reactions can be avoided. See sequences of the normal primers and the stop primers below:
  • Non-polymerase extendable primers are non-polymerase extendable primers:
  • Block primer 5' - 3' (SEQ ID NO: 20 with a stop group) : CTCCTGCTCATGATCCTACACCT-(STOP) 2D6*7/2D8P B, Block primer 5' - 3' (SEQ ID NO: 21 with a stop group) : ATGATCCTACGCCCGGATGT-(STOP)
  • the block primers By designing the stop primers (block primers) so they have an optimal annealing temperature a few 0 C higher than the normal polymerase extendable primers, the block primers will anneal to the pseudo gene sequences before the normal primers. It is however imperative that the blocking primers are designed so the mismatch of the blocking primer on the normal primer site, leaves the blocking primer with a few 0 C lower annealing temperature than the optimal annealing temperature of the normal primers. This way the annealing of the normal primer to the desired amplified sequence is not affected by the blocking primers.
  • the WT PCR setup comprises of the 2D6*7 F WT primer, the 2D6*7 R primer, the 2D6*7/2D7 B primer and the 2D6*7/2D8P B primer.
  • the MT PCR setup comprises of the 2D6*7 F MT primer, the 2D6*7 R primer, the 2D6*7/2D7 B primer and the 2D6*7/2D8P B primer.
  • nucleic acid sample is from a homozygote WT carrier, amplification will only occur in the WT PCR and not in the MT PCR. If the nucleic acid sample is from a homozygote MT carrier, amplification will not occur in the WT PCR but only in the MT PCR. Heterozygote carriers are identified by amplification in both the WT PCR and MT PCR.
  • the blocking primers are essential in this setup, as the 2D6*7 F WT primer matches sequences in the CYP2D7 and CYP2D8P genes, see below: 5'-...TGGCCTGGGGCCTCCTGCTCATGATCCTACACCTGGATGTGCAG...-3 ' CYP2D7 sequence
  • reaction mixes were prepared, a WT mix, a MT mix, a WT-S mix and a MT-S mix.
  • the WT and the MT mixes were the ASPC containing the stop primer while the WT-S and the MT-S were the ordinary ASA mixes without the stop primer.
  • the WT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 PI RWTn reverse primer and 2C19*2 PI RMTn+T 3'P stop primer.
  • the MT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 PI RMTn+T reverse primer and 2C19*2 PI RWTn 3'P stop primer.
  • the WT-S mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and the 2C19*2 PI RWTn reverse primer.
  • the MT-S mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and 2C19*2 PI RMTn+T reverse primer. All primers were HPLC purified and delivered by TAG Copenhagen A/S (Denmark).
  • the DNA template used in all PCRs was human genomic DNA previously genotyped as either homozygote WT (sample id : 119, wt/wt) homozygote MT (sample id : 149, mt/mt) regarding the CYP2C19*2 allele. See table 1 below for PCR setup.
  • One 10 ⁇ l reaction of each mix were prepared and thermal cycled by first an initial denaturing step of 15 seconds at 95° C, followed by 15 cycles of 1 second at 95° C then 1 second at 60° C and then 2 seconds at 72° C followed by 20 cycles of 1 second at 95° C then 1 second at 60° C where the annealing temperature was lowered 0.1° C per cycle and then 2 seconds at 72° C followed by a final extension step of 10 seconds at 72° C.
  • the annealing temperature in the last cycle (cycle no 35) is 58° C.
  • the thermal cycling was performed on a Primus 25 advanced thermal cycler (PEQLAB Biotechnologie GmbH, Germany).
  • PCRs were tested by gel electrophoresis using an in-house fabricated MicroGel electrophoresis setup.
  • a 2.5% agarose gel prepared in 0.5 X TBE buffer and stained with ethidium bromide (1.5 ⁇ l ethidum bromide in alO ml agarose gel solution) were cast with the dimensions of 30x54x3 mm (width x length x thickness).
  • the gel were covered in 1 X TBE buffer and loaded with 1 ⁇ l sample or DNA reference ladder pr. lane, and run at 35 V for 45 minutes. After 45 min. the gel was exposed with UV-light and an image of the gel was taken, see figure 4.
  • ASA PCRs PCRs no 3, 4, 7 and 8 amplification should only occur in PCR no 3 where the WT-S primer set perfectly matches the homozygote WT template and in PCR no 8 where MT-S primer set perfectly matches the homozygote MT template.
  • PCR no 7 which should not occur as the WT- S primer set only should provide amplification from the homozygote WT template and not from the homozygote MT template.
  • ASPC PCRs PCRs no 1, 2, 5 and 6
  • amplification should only occur in PCR no 1 where the WT primer set perfectly matches the homozygote WT template and in PCR no 6 where MT primer set perfectly matches the homozygote MT template. This is also observed on figure 4 where amplification is only observed in PCRs no 1 and 6 and not in PCRs no 2 and 5.
  • the PCRs were this time performed as real- timer PCR (qPCR) so amplification could be monitored for each cycle.
  • qPCR real- timer PCR
  • the idea is that the tag primers will produce a template to which the tag primer has a higher annealing temperature so the affinity of the tag primer to the template will increase during the cycles as more template with the tag part included is produced. It is speculated that inclusion of the stop primer is imperative for the specificity of the assay as the risk of unspecific amplification increases by introducing the tag primers. See tag primer sequences below.
  • the 5'-end tag part of the normal primer is underlined.
  • the tag part was designed so both the 2C19*2 RWTn tag primer and the 2C19*2 RMTn+T tag primer would form a template with a homogenous annealing temperature for the tagged primers.
  • reaction mixes were prepared, a WT mix, a MT mix, a WT-S mix and a MT-S mix.
  • the WT and the MT mixes are the ASPC containing the stop primer while the WT-S and the MT- S are the ordinary ASA mixes without the stop primer. All four mixes contain a version of the tagged reverse primers instead of a normal reverse primer.
  • the WT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 RWTn tag reverse primer and 2C19*2 PI RMTn+T 3'P stop primer.
  • the MT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 RMTn+T tag reverse primer and 2C19*2 PI RWTn 3'P stop primer.
  • the WT-S mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and the 2C19*2 RWTn tag reverse primer.
  • the MT-S mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer and 2C19*2 RMTn+T tag reverse primer. All primers were HPLC purified and delivered by TAG Copenhagen A/S (Denmark).
  • the amplification curves from the ASPC PCRs seen on figure 6 shows high amplification level in the WT PCR and low (equal to the level of the negative controls) amplification level in the MT PCR.
  • the amplification yield in the WT PCR is a factor 5 higher than the MT PCR amplification yield which correlates perfectly with the fact that only the WT allelic variant was present in the template DNA hence amplification should only have occurred in the WT PCR.
  • including the stop primer in the tagged normal primer assay setup provides the necessary specificity to discriminate between allelic variants.
  • the addition of the tag to the normal primer provides a high level of specific amplification which makes the setup suited for end- point read-out without the use of a TD PCR thermal cycling profile.
  • the MT mix contained a final primer concentration of 0.5 ⁇ M of both 2C19*2 PI Fn forward primer, 2C19*2 PI RMTn+T reverse primer and 2C19*2 PI RWTn 3'P stop primer. All primers were HPLC purified and delivered by TAG Copenhagen A/S (Denmark).
  • the DNA template used in all PCRs was human genomic DNA previously genotyped as either homozygote WT (sample id : 138, wt/wt), heterozygote (sample id: 140, wt/mt) or homozygote MT (sample id: 139, mt/mt) regarding the CYP2C19*2 allele. See table 3 for PCR setup.
  • PCRs were again tested by gel electrophoresis using an in-house fabricated MicroGel electrophoresis setup.
  • a 2.5% agarose gel prepared in 0.5 X TBE buffer and stained with ethidium bromide (1.5 ⁇ l ethidum bromide in a 10 ml agarose gel solution) were cast with the dimensions of 30x54x3 mm (width x length x thickness).
  • the gel were covered in 1 X TBE buffer and loaded with 1 ⁇ l sample or DNA reference ladder pr. lane, and run at 35 V for 45 minutes. After 45 minutes the gel was exposed with UV-light and an image of the gel was taken, see figure 7.
  • amplification should only have occurred in PCRs no 1, 3, 4 and 6.
  • PCRs no 1 and 6 amplification should have occurred as the normal primer perfectly matched the template DNA.
  • PCRs no 3 and 4 both allelic variants was present in the template DNA so amplification should have occurred as the normal primer perfectly matched the template DNA in both the WT and the MT reaction.
  • PCRs no 2 and 5 amplification should not have occurred as the STOP primer perfectly matched the template DNA hence preventing annealing and subsequently extension of the normal primer. From figure 7 amplification was observed in PCRs no 1, 3, 4, and 6 indicating that the ASPC PCR assay is well suited for SNP genotyping also in GC-rich regions. As the ASPC PCR assay setup both provides high specificity and high yield, the method also seems ideal for end-point read-out setups. No false positive reactions or amplification in the negative controls were observed.

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Abstract

La présente invention concerne un procédé qui permet d'accroître les caractéristiques de fidélité du produit d'une technique d'amplification spécifique d'allèle (ASA). Le procédé se fonde sur la 'concurrence entre les amorces' et aide à réduire au minimum la quantité de produit provenant d'une amplification non spécifique pouvant apparaître au cours d'une amplification ASA normale, lorsque la matrice interrogée contient une séquence mutée. Une amorce supplémentaire est incorporée dans la technique d'amplification enzymatique, ladite amorce possédant une correspondance avec la séquence mutée. L'amorce supplémentaire comprend en plus une extrémité-3' modifiée qui empêche l'extension.
PCT/DK2007/050178 2006-11-27 2007-11-27 Procédé d'amplification spécifique d'allèle à fidélité accrue WO2008064687A1 (fr)

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DE102013204712A1 (de) * 2013-03-18 2014-09-18 Siemens Aktiengesellschaft Verfahren und Kit zum sequenzspezifischen Stoppen einer Synthese eines zu einer einzelsträngigen Matrizen-Nukleinsäure komplementären Nukleinsäure-Strangs durch eine Polymerase
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WO2009043112A1 (fr) * 2007-10-04 2009-04-09 Commonwealth Scientific And Industrial Research Organisation Amplification d'acide nucléique
US8455197B2 (en) 2007-10-04 2013-06-04 Commonwealth Scientific And Industrial Research Organisation Nucleic acid amplification
EP2722399A1 (fr) * 2012-10-18 2014-04-23 Roche Diagniostics GmbH Procédé de prévention de produits à poids moléculaire élevé lors de l'amplification
EP2722403A1 (fr) 2012-10-18 2014-04-23 Roche Diagniostics GmbH Procédé de prévention de produits à poids moléculaire élevé lors de l'amplification
US9447476B2 (en) 2012-10-18 2016-09-20 Roche Molecular Systems, Inc. Method for preventing high molecular weight products during amplification
DE102013204712A1 (de) * 2013-03-18 2014-09-18 Siemens Aktiengesellschaft Verfahren und Kit zum sequenzspezifischen Stoppen einer Synthese eines zu einer einzelsträngigen Matrizen-Nukleinsäure komplementären Nukleinsäure-Strangs durch eine Polymerase
CN108138226A (zh) * 2015-10-18 2018-06-08 阿费梅特里克斯公司 单核苷酸多态性和插入缺失的多等位基因基因分型
WO2017070096A1 (fr) * 2015-10-18 2017-04-27 Affymetrix, Inc. Génotypage multiallèles de polymorphismes mononucléotidiques et d'insertions-délétions
JP2019500706A (ja) * 2015-10-18 2019-01-10 アフィメトリックス インコーポレイテッド 一塩基多型及びインデルの複対立遺伝子遺伝子型決定
RU2706203C1 (ru) * 2015-10-18 2019-11-14 Эффиметрикс, Инк. Мультиаллельное генотипирование однонуклеотидных полиморфизмов и индел-мутаций
CN108138226B (zh) * 2015-10-18 2022-02-11 阿费梅特里克斯公司 单核苷酸多态性和插入缺失的多等位基因基因分型
IL258795B (en) * 2015-10-18 2022-10-01 Affymetrix Inc Multiallelic genotyping of single nucleotide polymorphisms and indels
IL258795B2 (en) * 2015-10-18 2023-02-01 Affymetrix Inc Multiallelic genotyping of single nucleotide polymorphisms and indels
WO2018024562A1 (fr) * 2016-08-02 2018-02-08 Roche Diagnostics Gmbh Oligonucléotide auxiliaire pour améliorer l'efficacité de l'amplification et de la détection/quantification d'acides nucléiques
CN109642253A (zh) * 2016-08-02 2019-04-16 豪夫迈·罗氏有限公司 用于提高核酸的扩增和检测/定量的效率的辅助寡核苷酸
JP2019524123A (ja) * 2016-08-02 2019-09-05 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 核酸の増幅及び検出／定量の効率を改良するためのヘルパーオリゴヌクレオチド
US10443095B2 (en) 2016-08-02 2019-10-15 Roche Molecular Systems, Inc. Helper oligonucleotide for improved efficiency of amplification and detection/quantitation of nucleic acids
JP6999645B2 (ja) 2016-08-02 2022-02-04 エフ．ホフマン－ラ ロシュ アーゲー 核酸の増幅及び検出／定量の効率を改良するためのヘルパーオリゴヌクレオチド

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