WO2013093530A1 - Procédé de détermination de la séquence d'acides nucléiques fragmentés - Google Patents

Procédé de détermination de la séquence d'acides nucléiques fragmentés Download PDF

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WO2013093530A1
WO2013093530A1 PCT/HU2012/000137 HU2012000137W WO2013093530A1 WO 2013093530 A1 WO2013093530 A1 WO 2013093530A1 HU 2012000137 W HU2012000137 W HU 2012000137W WO 2013093530 A1 WO2013093530 A1 WO 2013093530A1
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pcr
segment
spacer
primer
sequence
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István PETÁK
Ferenc PINTÉR
Richárd CJSCHWAB
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Kps Orvosi Biotechnológiai És Egészségügyi Szolgáltató Kft.
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Publication of WO2013093530A1 publication Critical patent/WO2013093530A1/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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to field of molecular diagnostics. More specifically, the invention relates to sequencing methods for the efficient and reliable identification of the nucleotide sequence of damaged (fragmented or degraded) nucleic acids, primarily in biological samples. Furthermore, the present invention relates to the spacer molecules used in such methods and to the kits required for carrying out such methods.
  • the only biological samples available for the determination of the nucleotide sequence of nucleic acids carrying genetic information i.e., of DNA and RNA molecules, contain these nucleic acids in a fragmented form.
  • the most common examples include tumorous tissue samples removed for diagnostic purposes, fixed with formalin and stored embedded in paraffin. It is widely known that fragmentation of the nucleic acid contents of samples is inevitable already when fixing and storing the samples.
  • the necessity for determining the sequence of certain target regions of the nucleic acids extracted from such samples is increasing. However, due to their small sizes and low quantities, the detection of these nucleic acids is problematic during a subsequent analysis.
  • the most specific method for determining the sequence of nucleic acids involves PCR amplification of the target sequence followed by bidirectional dideoxy sequencing (Sanger's dideoxy sequencing or chain termination method).
  • PCR primer elongated with an overhanging M13 binding site has a greater tendency to dimer formation, which reduces PCR efficacy; in addition, during primer synthesis, primer synthesis errors, e.g., deletions and insertions occur more frequently, and this makes the sequence picture noisy and thereby reduce the sensitivity of the analysis.
  • each target sequence is amplified using separate primers with overhangs that overlap with the other target sequence and with the amplifying primer; next, the reaction products of the first PCRs are mixed and fused in a second PCR.
  • the fusion product emerges, the following problems are encountered: a) the PCR amplification of each target sequence in the mixture continues individually and in competition with the fusion product, b) the use of long primers reduces the efficiency of the PCR technique, c) the overlapping primers form dimers during the fusion PCR causing sequence noise, d) synthesis errors in the primers also appear as noise during sequence analysis, e) due to the two separate PCR reactions, the total costs are not reduced either.
  • WO 2007/025340 (Keith S. et al.) describes a method for the noise- and discrimination-free amplification of sequences comprising two amplification steps. This method is also useful for the random pre-amplification of DNA samples available in small quantities. Such increased sample quantities are very useful for the evaluation of more than one target sequences in subsequent analyses. However, this technique only increases the amount of the fragments but no their size. Therefore, very short DNA sequences will continue to fail to participate in the sequencing reaction.
  • Another common method for solving the above problem is to use primers that overhang the target sequence during the PCR amplification, which generates longer PCR products from shorter target sequences.
  • One disadvantage of this method is the ⁇ 0 fact that many primers will contain a few nucleotide errors - nucleotide deletions in several cases - as a result of synthesizing long primers. The sequence generated from this DNA strand will give a result which is shifted by one nucleotide. Upon superimposition, such shifted target sequences will render the sequence picture analytically noisy ("sequence noise").
  • One solution to this problem may be to attach a DNA molecule of at least 70 bp to the target sequence after the amplification.
  • OEP overlap extension PCR
  • the 3' end and 5' end of the linker primers used in the first PCR are complementary to the template DNA and to the adjacent DNA, respectively.
  • One disadvantage of this method is that the 5' end complementary to the adjacent DNA greatly diminishes primer efficiency during the amplification of the template sequence, and this in turn
  • primer dimers are formed during the first PCR, and this significantly reduces PCR efficiency in the case of small DNA sample amounts. Unless they are removed, such primer dimers will fuse during the second PCR in the same way as the DNA strand generated from the template sequence. Such DNA strands with no template content generate a noise during sequencing.5 From the method, it also follows that the sequence differences in the linker primers will be incorporated into the fused strand. In case of an insertion or deletion, this will appear as noise running through the entire sequencing electropherogram with an amplitude corresponding to the proportion of primers comprising the insertion/deletion.
  • the above disadvantages are major obstacles in applying the
  • the problem to be solved by the present invention is to Increase the efficiency and reliability of the dideoxy sequencing of individual sequences in samples containing fragmented nucleic acids by overcoming the disadvantages of the above methods.
  • a technique based on overlap extension PCR was designed, which ensures the fusion of short PCR products with the shortest possible primers; in addition, a mutation-free adapter sequence was designed, which fills up the noisy sequence generated during the first 30 to 50 bp of the sequencing reaction without the need for amplifying long PCR products.
  • a single PCR is used to attach previously prepared long artificial spacer DNA molecules to both ends of the target sequence to be analysed, and this allows the target sequence to be localised at a sufficient distance from the binding site of the sequencing primer to ensure noise-free Sanger reading.
  • the method allows bidirectional Sanger sequencing to read the same target sequence from a shorter DNA molecule than the conventional method without fusion.
  • this is achieved by eliminating the need for adding sequences that are complementary with other DNA strands to the DNA strand comprising the target sequence before the fusion, that is, the need for using primers that contain sequences other than those complementary to the template DNA as this reduces the efficiency/
  • Another particular advantage is that the adapter DNA hybridises at the binding site of the primer used for the amplification of the target sequence or a few nucleotides more inside, and this prevents adapters to attach to the primer dimers generated by the previous amplification. Therefore, this method is even more advantageous than when ligation is used to attach the adapter sequences to the target sequences.
  • Figure 1 Determination of the sequence of nucleic acids by fusing spacer molecules
  • Figure 2 Structure of the spacer molecule.
  • Figure 3 Location of the hybridisation site of the primer used in the pre-fusion PCR and of the spacer molecule.
  • Figure 4 Key features of the method used to analyse Nl resistance mutations in H1 N1 influenza. The codons of the most important mutations were marked in accordance with the nomenclature of the H3N2 virus.
  • Figure 5 Efficient functioning of the method analysing mutations (segments in bold on the electropherogram) causing the Nl resistance of H1 N1 from the period between 1977 and 2008.
  • Bold italic letters sequences corresponding or complementary to the primers of the conventional PCR; letters in frames: sequences corresponding or complementary to the primers of the nested PCR.
  • New method grey marking: sequences corresponding to the forward primer and complementary to the reverse primer of the pre-fusion PCR; underlining: sequences corresponding to the template-specific segment of the forward spacer molecule and complementary to the template-specific segment of the reverse spacer molecule; white letters on a black background: sites of the most significant mutations of Exon 21 of EGFR.
  • a single PCR is used to attach previously synthesised spacer DNA molecules to both ends of the target sequence to be analysed with a length that allows the target sequence to be localised at a sufficient distance from the 3' end of the primer to ensure noise-free Sanger reading.
  • amplification before the dideoxy sequencing is also possible from a nucleic acid longer only by 2x20 bp (2* the length of the primer) than the target sequence.
  • the target sequence can be PCR amplified before the PCR used for coupling the spacer molecule in order to ensure that the template is available in sufficient number of copies. This can be achieved by using primers that only contain sequences complementary to the template. In such cases, amplification is also possible from a nucleic acid longer by 2x30 bp (2* the length of the primer + 2> ⁇ 10) than the target sequence.
  • the spacer molecule used in the method of the invention which may be single- or double-stranded DNA, comprises the following (see Figure 2):
  • one aspect of the invention provides a method for the determination of the sequence of fragmented nucleic acids by the PCR amplification of a target sequence and the subsequent dideoxy sequencing, comprising the steps of:
  • the nucleic acid is isolated from the biological sample.
  • step a) isolation
  • cells containing the nucleic acid are isolated from the biological sample, preferably by microdissection.
  • the PCR reaction fusing the spacer DNA molecules is presented in Figure 1 :
  • the complementary 3' ends of the spacer DNA molecule and of the DNA molecules comprising the target sequence hybridise, and thus the polymerase generates the second strand of the DNA molecule fused from one end. Simultaneously, the same occurs on the other end of the target sequence.
  • the spacer DNA molecule for the other end hybridises to the singly fused DNA molecule in the same way giving rise to the double-stranded DNA molecule fused at both ends, which comprises sequences complementary to both the forward and the reverse primer, thus also allowing the amplification of the fused molecule.
  • the PCR reaction in this case is a multiplex reaction in which fusion and amplification occurs in the same step.
  • this reaction should be designed to ensure that the 3' ends of the primers are complementary to the sequences of the template nucleic acid located at least 0 to 70 bp closer to the 3' end than the template specific section of the spacer DNA molecule (see Figure 3). This prevents fusion between the primer dimers generated in the first PCR and the spacer DNA molecules during the fusion PCR.
  • another aspect of the invention provides a method for the determination of the sequence of fragmented nucleic acids by a two-step PCR amplification of a target sequence and the subsequent dideoxy sequencing comprising the steps of:
  • the nucleic acid is isolated from the biological sample.
  • step a) isolation
  • cells containing the nucleic acid are isolated from the biological sample, preferably by microdissection.
  • Step b) a further nested PCR may be performed after the first PCR, if required.
  • the nucleic acid to be sequenced may be DNA or RNA.
  • fragmented nucleic acid to be sequenced means fragmented DNA, RNA or miRNA.
  • fragmented nucleic acid means a nucleic acid with a length of 30 to 1000 bp, preferably 55 to 300 bp.
  • the method can be advantageously used in any application in which the DNA segment to be sequenced is not part of a DNA molecule which is at least 70 bp longer than the target segment.
  • the sequence of a DNA or RNA segment of 100 bp is to be determined, then it should be located in the middle of a DNA segment of at least 240 bp in length to allow sequencing using the known methods.
  • a segment of 200 bp to be sequenced should be located in the middle of a segment of at least 340 bp in length.
  • the upper limit is only represented by the technical limit of the dideoxy sequencing, which is approx. 1000 bp.
  • Bio samples include biopsies, tissue samples, cytology smears, blood samples, ascites fluid, spinal chord, urine, etc.
  • tissue samples are preferred.
  • the nucleic acid sample may be taken from human, animal or plant tissues or cells.
  • Application of the method in case of a biological sample containing nucleic acid fragments of small sizes and in low amounts is particularly preferable. In such cases, the number of DNA fragments available as templates are increased in proportion to the reduction in the size of the target sequence.
  • One example for such an application is the processing of histological samples or cytology smears in which laser microdissection microscopy is used to introduce nucleic acids corresponding to a few, or a few hundred or a few thousand cells into a test tube, and
  • nucleic acid is either isolated or directly amplified by PCR.
  • the spacer DNA molecule is always synthesised by the user or others commissioned by the user on the basis of the target sequence, which should be known in advance.
  • the spacer molecule may comprise a binding site for the amplification primer, an M13 binding site and a binding site
  • forward and reverse spacer molecules are used and these can be prepared in the following manner.
  • a PCR is performed in which spacer molecules i0 are fused to the target sequences.
  • the reaction mixture should contain the following components in addition to those required for the functioning of the polymerase: spacer molecules fusing to the ends of the target sequence, and in lower amounts than the amplification primers; and forward and reverse primers amplifying the fused molecule.
  • fusion is performed on the amplicons thus prepared.
  • spacer molecules are fused to both ends of the amplicons (see Examples 1 to 3), therefore Figure 1 applies to such systems.
  • fusion only occurs on one end (see Figure 4), and the other end is used for overlap fusion with sequences overlapping between the 2 neuraminidase gene regions, which are prepared by primers overhanging into the other region during the pre- fusion PCR amplification (see Examples 4 and 5).
  • the product obtained during the fusion and amplification PCR is sequenced directly or upon purification.
  • the PCR product can be purified in known manners, e.g., by ethanol precipitation, on silica columns with the use of XTerminator®.
  • Sequencing is performed using a method known perse.
  • the method of the invention can be used in many fields.
  • One field of application is the identification of gene sequences in plant, animal or human tissue samples containing fragmented DNA.
  • the method of the invention can be used to detect mutations in the H1 N1 influenza neuraminidase gene, and this may serve as the basis for therapy planning.
  • the invention is useful for determining the nucleotide sequence of fragmented nucleic acids which optionally contain mutations and these mutations can be reliably detected by precise sequencing.
  • One typical field of this is detecting mutations in fragmented DNA samples from tumorous tissues.
  • the sequencing method of the invention is particularly useful for the reliable analysis of tissue samples fixed and stored in formalin and thus containing damaged, fragmented nucleic acids.
  • One typical field of application of this is cancer diagnostics, i.e., the identification of genetic markers affecting treatment strategies.
  • Non-limiting examples for this include known mutations in Exons 18, 19, 20 and 21 of EGFR, mutations in Exons 2, 3 and 4 of KRAS, HRAS and NRAS, mutations in Exons 1 1 and 15 of BRAF, mutations in Exons 19 and 20 of HER2, mutations in Exons 1 , 9 and 20 of the PI3KCA gene, mutations in Exons 9, 1 1 , 13 and 17 of KIT, mutations in Exons 12, 14 and 18 of PDGFR-A and mutations in Exons 20, 22, 23, 24 and 25 of the ALK gene.
  • kits for carrying out the methods of the invention may contain the following components for the method of the invention using a single PCR: one or more spacer molecules; or one or more spacer molecules and amplification primers. If desired, the kit may also contain the polymerase and reagents required for the PCR.
  • the kit may contain the following components for the method of the invention using more than one PCR: one or more spacer molecules; or one or more spacer molecules and amplification primers for the fusion PCR; or one or more spacer molecules, amplification primers for the fusion PCR and amplification primers for the first amplification PCR. If desired, the kit may also contain the polymerases and reagents required for the PCR reactions.
  • the method of the invention is illustrated with the amplification and sequencing of 5 nucleic acid sequences.
  • the objective was to analyse human gene segments. Due to the mutations present in them, we wished to analyse the most relevant regions of Exon 2 of KRAS and Exons 19 and 21 of EGFR.
  • the initial nucleic acid was DNA.
  • the additional two gene segments analysed by the method of the invention were two segments of the influenza neuraminidase gene which contain mutations associated with varying degrees of resistance against various neuraminidase inhibitors. Since an RNA virus is concerned, RNA is used as template in this analysis.
  • the starting materials included gene sequences of H1 N1 influenza viruses from the period between 1977 and 2005.
  • the assay thus established can detect all influenzas from the period between 1977 and 2008.
  • an optimised assay was devised for the detection of the new type H1 N1 influenza, in which the primers and the template-specific parts of the spacer contain a few nucleotides that are different from the components of the H1 N1 assay for the year 2008.
  • Table 1 Parameters of the diagnostic systems based on the method of the invention.
  • influenza the 10- to 11 -bp sequence marked with an asterisk does not amplify from the template but fuses with the primers containing sequences overhanging into the other gene region.
  • Preparation of the spacer molecules For the diagnostic system, one forward and one reverse spacer molecule was prepared. The parts of the framework of the forward spacer molecules which are around the pCR2.1-TOPO vector (Invitrogen) M13F primer, and the parts of the framework of the reverse spacer molecules which are around the pENTR/H1/TO vector (Invitrogen) M13R primer were used.
  • sequence acting as the framework of the pCR2.1-TOPO vector forward spacer molecules set forth as SEQ ID NO:1 (104 bp, the segment marked grey corresponds to the forward amplification primer, and the segment in white letters on a black background corresponds to the sequence of the M13F sequencing primer):
  • sequence acting as the framework of the pENTR/H1/TO vector reverse spacer molecules set forth as SEQ ID NO: 2 (1 10 bp, the segment marked grey corresponds to the reverse amplification primer, and the segment in white letters on a black background corresponds to the sequence of the M13R primer):
  • reverse primer SEQ ID NO: 4
  • grey marking restriction enzyme binding site, underlining: a segment complementary to the KRAS gene segment, italic letters: a sequence identical to the pENTR/H1/TO vector segment
  • the attachment of the forward and reverse spacer molecules were achieved in separate PCR reactions. The only difference between the two reactions was in the primers used. Amplification was carried out in solutions containing the pCR2.1-TOPO vector and the pENTR/H1/TO vector, respectively, using AccuPrime Taq HiFi DNS polymerase (Invitrogen, 0.02 U/ ⁇ ), 1x AccuPrime I PCR buffer and forward and reverse primers each at a concentration of 0.4 ⁇ . The PCR included the following cycles:
  • Cycle 5 (1x) Step 1 4°C -
  • the artificial sequences thus generated were cloned using the CloneJET1.2 PCR Cloning Kit (Fermentas) and Top10 bacteria (Invitrogen).
  • the pJET1.2 vectors containing the spacer molecules were isolated using MidiPrep.
  • the forward and the reverse spacer molecule were excised from the vector using the restriction enzymes Mlu Nl and Avill, respectively (both from Roche, 0.41 ) / ⁇ , after 16 hours of incubation at 37°C).
  • E-Gel SizeSelect Invitrogen
  • the sequence of the forward spacer molecule (SEQ ID NO: 7) generated by the restriction cleavage is as follows (137 bp, the segment marked grey corresponds to the forward amplification primer, the segment in white letters on a black background corresponds to the M13F sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the KRAS gene):
  • the sequence of the reverse spacer molecule (SEQ ID NO: 8) generated by the restriction cleavage is as follows (134 bp, the segment marked grey corresponds to the reverse amplification primer, the segment in white letters on a black background corresponds to the M13R sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the KRAS gene):
  • CTCTTGC Pre-fusion PCR amplification The samples containing fragmented DNA isolated from a formalin-fixed, paraffin-embedded sample were subjected to pre-fusion PCR amplification. The PCR conditions were identical with those of the reaction used in the preparation of the spacer molecule.
  • the sequence of the forward and reverse primers were GACATGTTCTAATATAGTCACATTTTCATTATT (SEQ ID NO: 9) and TCTGAATTAGCTGTATCGTCAAGG (SEQ ID NO:10), respectively.
  • the following 120-bp amplicon (SEQ ID NO:1 1 ) was generated upon the PCR (sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, underlining: sequences corresponding to the template-specific portion of the forward spacer molecule and complementary to the template-specific portion of the reverse spacer molecule, white letters on a black background: sites of the most significant KRAS mutations):
  • the forward and reverse spacer molecules were fused to the amplicon generated in the previous step using PCR.
  • the PCR solution contained the following components: AccuPrime Taq HiFi DNA polymerase (Invitrogen, 0.02 U/ ⁇ ), 1 * AccuPrime I PCR buffer, a forward primer (ATTAAGTTGGGTAACGCCAGGGTTT) (SEQ ID NO: 12) and a reverse primer (AGATTTTGAGACACGGGCCAGA) (SEQ ID NO:13) each at a concentration of 0.4 ⁇ , and forward and reverse spacer molecules.
  • the PCR solution contained ⁇ 0.012 pM forward and reverse spacers.
  • the PCR included the following cycles:
  • Step 3 68°C 1 m in OOsec
  • Cycle 4 (1x) Step 1 68°C 10min OOsec Cycle 5: (1x) Step 1 4°C ⁇
  • the fusion product was purified using the Exo-SAP-IT (USB) enzyme mixture, and a bidirectional termination reaction was carried out using the BigDye Terminator v3.1 Sequencing Kit (Applied 20 Biosystems), the M13F primer (TGTAAAACGACGGCCAGT) (SEQ ID NO:15), and the M13R primer (CAGGAAACAGCTATGAC) (SEQ ID NO:16).
  • the product of the termination reaction was purified using the BigDye XTerminator kit (Applied Biosystems), and the DNA sequence was read by capillary electrophoresis performed in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems).
  • the spacer molecules For the diagnostic system, one forward and one reverse spacer molecule was prepared as in Example 1 with the following modifications.
  • the following reverse primer (SEQ ID ⁇ . ⁇ 7) was used in the OEP reaction to attach the segment complementary with the EGFR gene downstream of the M13F primer and the additional nucleotides required for the restriction cleavage to the pCR2.1-TOPO vector sequence (grey marking: restriction enzyme binding site, underlining: a sequence identical to the EGFR gene segment, italic letters: a segment complementary to the vector sequence):
  • reverse primer SEQ ID NO: 18
  • grey marking restriction enzyme binding site, underlining: a segment complementary to the EGFR gene segment, italic letters: a sequence identical to the pENTR/H1/TO vector segment
  • a guanine in the pENTR/ ⁇ ⁇ vector was replaced by cytosine in the spacer molecule (white letters on a black background in the previous sequence) to eliminate the CAGCTG restriction cleavage site present in this segment in order to prevent a subsequent cleavage of the spacer molecule.
  • Hpal and Pvull were used, respectively, for the restriction cleavage.
  • the sequence of the forward spacer molecule (SEQ ID NO:21) generated by the restriction cleavage is as follows (131 bp, the segment marked grey corresponds to the forward amplification primer, the segment in white letters on a black background corresponds to the M13F sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the EGFR gene): AACGCGATT- PTJGGGTAACGGCAGSSTTFTCCCAGTCACGACGTJTGTAAAACGACGGCCA EHGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGTGGATCCCAGAAGGTGA
  • the sequence of the reverse spacer molecule (SEQ ID NO:22) generated by the restriction cleavage is as follows (133 bp, the segment marked grey corresponds to the reverse amplification primer, the segment in white letters on a black background corresponds to the M13R sequencing primer, the underlined segment corresponds to a sequence complementary to a portion of the EGFR gene, white italic letter on a black background: nucleotides replaced in the vector sequence during the OEP): CTGGTGCAATGTAACATCAGAGATTtTGAGACAt!GGGCCAG ⁇
  • Pre-fusion PCR amplifica tion The samples containing fragmented DNA isolated from a formalin-fixed, paraffin-embedded sample were subjected to pre-fusion PCR amplification. The only difference between this PCR and the reaction used in Example 1 was in the primers used.
  • the sequence of the forward and reverse primer were TCTCTGTCATAGGGACTCTGGA (SEQ ID NO:23) and AGCCATGGACCCCCACAC (SEQ ID NO:24), respectively.
  • sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, underlining: sequences corresponding to the template-specific portion of the forward spacer molecule and complementary to the template-specific portion of the reverse spacer molecule, white letters on a black background: sites affected by the most significant mutations of Exon 19 of EGFR):
  • the spacer molecules For the diagnostic system, one forward and one reverse spacer molecule was prepared as in Example 1 with the following modifications.
  • the following reverse primer (SEQ ID NO:27) was used in the OEP reaction to attach the segment complementary with the EGFR gene downstream of the M13F primer and the additional nucleotides required for the restriction cleavage to the pCR2.1-TOPO vector sequence (grey marking: restriction enzyme binding site, underlining: a sequence identical to the EGFR gene segment, italic letters: a segment complementary to the vector sequence):
  • reverse primer SEQ ID NO:28
  • grey marking restriction enzyme binding site, underlining, a segment complementary to the EGFR gene segment, italic letters: a sequence identical to the pENTR/H1/TO vector segment
  • the following forward primer (SEQ ID NO:29) was used to introduce the restriction enzyme binding site on the other end of the pCR2.1-TOPO vector sequence (grey marking: restriction enzyme binding site, italic letters: a sequence identical to the vector segment):
  • the sequence of the forward spacer molecule (SEQ ID NO:31) generated by the restriction cleavage is as follows (131 bp, the segment marked grey corresponds to the forward amplification primer, the segment in white letters on a black background corresponds to the M13F sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the EGFR gene):
  • the sequence of the reverse spacer molecule (SEQ ID NO:32) generated by the restriction cleavage is as follows (127 bp, the segment marked grey corresponds to the reverse amplification primer, the segment in white letters on a black background corresponds to the M13R sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the EGFR gene):
  • Pre-fusion PCR amplification The samples containing fragmented DNA isolated from a formalin-fixed, paraffin-embedded sample were subjected to pre-fusion PCR amplification. The only difference between this PCR and the reaction used in Example 1 was in the primers used.
  • the sequence of the forward and reverse primer were GAAAACACCGCAGCATGTC (SEQ ID NO:33) and AAAGCCACCTCCTTACTTTGC (SEQ ID NO:34), respectively.
  • the following 107-bp amplicon (SEQ ID NO:35) was generated upon the PCR (sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, underlining: sequences corresponding to the template-specific portion of the forward spacer molecule and complementary to the template-specific portion of the reverse spader molecule, white letters on a black background: sites of the most significant mutations of Exon 21 of EGFR):
  • TGCAGAAGGAGGCAAAGTTGCC ATCCAGCTGATATCCCCTATAGTGAGTCGTATTACBDMMRI ⁇
  • the starting materials included gene sequences of H1 1 influenza viruses from the period between 1977 and 2005.
  • the assay thus established can detect all influenzas from the period between 1977 and 2008.
  • spacers were fused to both ends of the amplicons (thus, Figure 1 applies to these systems)
  • spacers were only fused to one end (as illustrated in Figure 4). The other end was subjected to overlap fusion with overlapping sequences between the 2 neuraminidase gene regions generated by primers overhanging into the other region in the pre-fusion PCR amplification.
  • spacer molecule For the diagnostic system, one forward spacer molecule was prepared as in Example 1 with the following modifications.
  • the following reverse primer (SEQ ID NO:37) was used in the OEP reaction to attach the segment complementary with the NA gene downstream of the M13F primer and the additional nucleotides required for the restriction cleavage to the pCR2.1-TOPO vector sequence (grey marking: restriction enzyme binding site, underlining: a sequence identical to the NA gene segment, italic letters: a segment complementary to the vector sequence):
  • the following forward primer (SEQ ID NO:38) was used to introduce the restriction enzyme binding site on the other end of the pCR2.1-TOPO vector sequence (grey marking: restriction enzyme binding site, italic letters: a sequence identical to the vector segment):
  • Dral was used for the restriction cleavage.
  • the ⁇ 6pg (4 wells) spacer molecule was individually collected into the 1 ml aqueous solution.
  • sequence of the spacer molecule (SEQ ID NO:39) generated by the restriction cleavage is as follows (128 bp, the segment marked grey corresponds to the forward 25 Pffl/HlOTtfWn amplification primer, the segment in white letters on a black background corresponds to the M13F sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the NA gene):
  • RNA pre-fusion PCR amplification Upon isolation of the RNA, RT-PCR was used. Using Qiagen's OneStep RT-PCR Kit, the reaction solution contained 1 * buffer, 400 ⁇ of each dNTP, 0.04 ⁇ / ⁇ enzyme mix, 0.6 ⁇ forward and reverse primer, and 0.1 U/ ⁇ RNasin Plus RNAse Inhibitor (Promega). The RT-PCR included the following cycles:
  • the two NA gene regions were amplified in separate reactions. During the amplification of the first gene region, the following primers were used:
  • the following 206-bp amplicon (SEQ ID NO:44) was generated upon amplifying the first gene region (using the RNA of the Solomon Islands/3/2006 virus as template; sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, in bold italic letters: a segment complementary to the other gene region; underlining: sequences corresponding to the template- specific portion of the spacer molecule, white letters on a black background: sites of the most significant mutations causing resistance to neuraminidase inhibitors): AAGACAACAGCAT AGAATTGGCTC C AAAG GAG AT GT T T T T GT CATAAGA— C C T T T T C AT A
  • the following 196-bp amplicon (SEQ ID NO.45) was generated upon amplifying the second gene region (using the RNA of the Solomon Islands/3/2006 virus as template; sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, in bold italic letters: a segment complementary to the other gene region; white letters on a black background: sites of the most significant mutations causing resistance to neuraminidase inhibitors):
  • the PCR solution for the fusion contained AccuPrime Taq HiFi DNA polymerase (Invitrogen, 0.02 U/ ⁇ ), 1 x AccuPrime I PCR buffer, a forward primer (SEQ ID NO:46) (ATTAAGTTGGGTAACGCCAGGGTTT) and a reverse primer (SEQ ID NO:47) (CACTCCACTGCAGATGTATCCTATTT) each at a concentration of 0.4 ⁇ .
  • One fifth of the reaction volume was from the 100* dilution of the PCR solution containing the amplicons of one gene region, another fifth was from the 100* dilution of the PCR solution containing the amplicons of the other gene region, and the third fifth was from the gel isolate of the spacer molecules.
  • samples ATL and AT are Fluval AB es Fluval P injections (Omnivest), respectively; these are purified, concentrated and formalin-inactivated suspensions of three influenza strains and a single strain, respectively, all propagated in embryonated hen's eggs.
  • Fluval AB contains a H1 N1 from 2007 (A/Brisbane/59/2007), a H3N2 from 2007 (A/Uruguay/716/2007) and an influenza B strain from 2008.
  • Fluval P contains a new type the H1 N1 influenza A virus (A/California/7/2009).
  • sequence of the spacer molecule (SEQ ID NO:50) generated by the restriction cleavage is as follows (128 bp, the segment marked grey corresponds to the forward amplification primer, the segment in white letters on a black background corresponds to the M13F sequencing primer, and the underlined segment corresponds to a sequence complementary to a portion of the NA gene):
  • Pre-fusion PCR amplification The only difference between the RT-PCR carried out on the RNA isolate and the reactions used in Example 1 was in the primers used.
  • AAAGACAACAGTATAAGAATCGGTTCCA SEQ ID NO:51
  • the following 207-bp amplicon (SEQ ID NO:55) was generated upon amplifying the first gene region (using the RNA of the California/07/2009 virus as template; sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, in bold italic letters: a segment complementary to the other gene region; underlining: sequences corresponding to the template- specific portion of the spacer molecule, white letters on a black background: sites of the most significant mutations causing resistance to neuraminidase inhibitors):
  • the following 196-bp amplicon (SEQ ID NO:56) was generated upon amplifying the. second gene region (using the RNA of the California/07/2009 virus as template; sequences with grey marking correspond to the forward primer and are complementary to the reverse primer, in bold italic letters: a segment complementary to the other gene region; white letters on a black background: sites of the most significant mutations causing resistance to neuraminidase inhibitors):
  • Sequencing of the fusion product The forward and reverse termination reactions were performed using the M13F primer and the reverse primer used during the fusion of the product, respectively. Other conditions of the sequence analysis were identical to those as described in Example .
  • the method analysing the neuraminidase gene regions of the new type H1 N1 was tested with the RNA isolated from the Fluval P vaccine (Omnivest).
  • the vaccine contains the new type virus A/California/7/2009 in an inactivated form.
  • the assay method was efficiently functioning in this case too ( Figure 6).
  • the virus-specific sequences of the fusion product were identical to the base sequence of the gene regions corresponding to the neuraminidase gene of A/California/7/2009 downloaded from the NCBI database.
  • Example 6 Reduced allele discrimination (allele bias) during the analysis of Exon 21 of the EGFR gene using the method of the invention
  • DNA was isolated from an FFPE sample with a tumour ratio of 30% according to the pathologist's opinion.
  • the sample was diluted to the operational limit of the conventional one-step PCR (10* dilution).
  • the diluted sample was subjected to 10 mutation analyses using a conventional method (a normal PCR followed by a nested PCR) and to another 10 using the method of the invention.

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Abstract

L'invention concerne de nouveaux procédés de séquençage pour l'identification efficace et fiable de la séquence nucléotidique d'acides nucléiques fragmentés ou dégradés, principalement dans des échantillons biologiques. De plus, la présente invention concerne des molécules espaceurs utilisées dans de tels procédés et les trousses nécessaires pour la mise en œuvre de tels procédés.
PCT/HU2012/000137 2011-12-20 2012-12-19 Procédé de détermination de la séquence d'acides nucléiques fragmentés WO2013093530A1 (fr)

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CN106755418A (zh) * 2016-12-24 2017-05-31 广州艾基生物技术有限公司 一种外显子组装测序方法
US10167509B2 (en) 2011-02-09 2019-01-01 Bio-Rad Laboratories, Inc. Analysis of nucleic acids

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10167509B2 (en) 2011-02-09 2019-01-01 Bio-Rad Laboratories, Inc. Analysis of nucleic acids
US11499181B2 (en) 2011-02-09 2022-11-15 Bio-Rad Laboratories, Inc. Analysis of nucleic acids
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CN105555972A (zh) * 2013-07-25 2016-05-04 伯乐生命医学产品有限公司 遗传测定
US9944998B2 (en) 2013-07-25 2018-04-17 Bio-Rad Laboratories, Inc. Genetic assays
CN105555972B (zh) * 2013-07-25 2020-07-31 伯乐生命医学产品有限公司 遗传测定
CN106755418A (zh) * 2016-12-24 2017-05-31 广州艾基生物技术有限公司 一种外显子组装测序方法
CN106755418B (zh) * 2016-12-24 2020-08-21 广州艾基生物技术有限公司 一种外显子组装测序方法

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