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WO2002034939A2 - Nouveau procede d'analyse genomique - Google Patents

Nouveau procede d'analyse genomique Download PDF

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Publication number
WO2002034939A2
WO2002034939A2 PCT/CA2001/001487 CA0101487W WO0234939A2 WO 2002034939 A2 WO2002034939 A2 WO 2002034939A2 CA 0101487 W CA0101487 W CA 0101487W WO 0234939 A2 WO0234939 A2 WO 0234939A2
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WIPO (PCT)
Prior art keywords
primer
dna
oligonucleotide
sequence
extension
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PCT/CA2001/001487
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English (en)
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WO2002034939A3 (fr
Inventor
Alex H. C. Yu
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Mbi Fermentas Inc.
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Priority to AU2002213695A priority Critical patent/AU2002213695A1/en
Publication of WO2002034939A2 publication Critical patent/WO2002034939A2/fr
Publication of WO2002034939A3 publication Critical patent/WO2002034939A3/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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • 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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention relates to the field of genomic analysis. More specifically, it relates to a process for generating extension primers by capturing sequences from DNA fragments and their use in genomic analysis, including in bacterial genotyping.
  • strain typing is often required as an integral part of epidemiological investigations.
  • the data generated from strain typing is used to identify outbreak-related strains and to distinguish epidemic from endemic or sporadic isolates.
  • traditional methods such as bacteriophage typing and serotyping, have been supplemented or replaced in many laboratories. This is driven by the need for a strain typing method that can be used to type a broader array of bacterial species.
  • bacteriophage typing and serotyping work very well in epidemiological studies of Staphylococcus aureus and Salmonella species, they are limited in their applications to different bacterial species.
  • PFGE pulsed-field gel electrophoresis
  • RAPD random amplified polymorphic DNA
  • PFGE analysis is essentially an improved method over the previously used method based on restriction fragment length polymorphism (RFLP).
  • genomic DNA is digested by a frequently cutting restriction endonuclease (RE) into hundreds of fragments. These fragments are separated by agarose gel electrophoresis and then transferred to a membrane. The DNA on the membrane is then hybridized with a specific DNA probe. The probe is so designed that only few fragments will hybridize to it. Variations in the number and size of the fragments detected by hybridization can be used to distinguish different bacterial strains.
  • Ribotyping the use of ribosomal RNA probe in RFLP, is one common typing method based on RFLP.
  • DNA insertion element IS6110 is currently the method of choice for typing isolates of My cobacterium tuberculosis based on RFLP.
  • An insertion element is a DNA fragment with a defined structure that is capable of moving independently and inserting in multiple locations in plasmids or chromosome. Since the insertion element is mobile, the number and the locations of the insertions vary greatly from strain to strain. This approach has been proven to be reliable and discriminatory for isolates with more than 5 copies of IS6110. However, the approach is still limited to this particular species.
  • PFGE became the method of choice in typing strains based on fragment patterns produced by cleaving chromosomal DNA with restriction endonucleases.
  • PFGE analysis the chromosomal DNA is cut with a restriction endonuclease that generates approximately 10 to 30 fragments.
  • PFGE is used to resolve all of these bands and the gel pattern generated can be used to type different bacterial strains. It is currently the method of choice for typing most species. Almost all bacterial species are typeable by PFGE analysis except C difficile, the DNA of which spontaneously degrades during cell lysis.
  • PCR ⁇ b ased “ methods” have “ bee developed " an " applied to identify bacterial strains and species.
  • specific PCR primers are used to amplify unique products from the bacterial DNA for diagnostic purposes.
  • multiple arbitrary amplicon profiling (MAAP) techniques use non-specific primers to target multiple and arbitrary amplicons as described by Caetano-Anolles, PCR Methods Applic. 3:85-94 (1993).
  • MAAP multiple arbitrary amplicon profiling
  • the variation in number and length of the amplified DNA fragments can be used to determine the r elatedness of bacterial strains. A number of variations of this approach has been developed.
  • Random amplified polymorphic DNA (RAPD) analysis is a MAAP techniques that differ in primer length, amplification stringency, and procedure used to resolve DNA patterns.
  • the DNA profiles obtained from these methods are distinct due to the difference in the sizes of the primers and the amplification conditions employed. For example, RAPD analysis resolves fewer amplification products than DAF.
  • RAPD is a random mdeterminate amphfication process that uses stochastic annealing events to randomly amplify DNA fragments.
  • known repetitive DNA motifs and highly conserved intergenic repetitive sequences have been exploited as primer binding sites for the amplification of bacterial DNA.
  • REP-PCR is a determinate process that targets multiple sites of known sequence but unknown locations, and amplifies DNA fragments between repetitive elements only.
  • repetitive sequences include the 33- to 40-bp repetitive extragenic palindromic (REP) elements discovered in the genomes of E. coli and S. typhimurium, the 124- to 127 bp enterobacterial repetitive intergenic consensus (ERIC) elements discovered in gram-negative bacteria species, and the 154-bp BOX elements discovered in the genome of S. pneumoniae. All these motifs are genetically stable and differ only in their copy numbers and chromosomal locations between species. Their copy numbers vary from approximately 25 copies for BOX, approximately 30 to 150 copies for ERIC, to approximately 500 to 1000 copies for REP.
  • REP repetitive extragenic palindromic
  • ERIC enterobacterial repetitive intergenic consensus
  • the invention relates to a method of . analyzing DNA which includes the step of ligating a first oligonucleotide and a DNA f agment to form a ligated product.
  • the oligonucleotide comprises a known sequence and a recognition site of a class LTS RE.
  • the ligated product is then digested with the class LTS RE to release a second oligonucleotide comprising the first oligonucleotide and a sequence from the DNA fragment.
  • a reaction mixture is formed comprising the DNA sample, the second oligonucleotide, a first amplification primer and a second amphfication primer under conditions suitable to drive extension and amphfication.
  • the first amplification primer specifically hybridizes to the known sequence and the second amplification primer specifically hybridizes to a sequence in the DNA fragment.
  • the presence of an extension product in the reaction mixture is then determined.
  • the invention also relates to a method of preparing an extension primer with captured genomic DNA sequences.
  • the method includes the steps of digesting genomic DNA of a species to generate DNA fragments and ligating the resulting DNA fragments with a first oligonucleotide to form ligated products.
  • the oligonucleotide comprises a known sequence and a recognition site for a class ES RE.
  • One or more ligated products are then amplified with a first and second primer.
  • the first primer specifically hybridizes to the known sequence and the second primer specifically hybridizes to a sequence in the genomic DNA.
  • the one or more amplified product is then digested with the class HS RE to generate an extension primer comprising the first oligonucleotide and a sequence from the genomic DNA.
  • the invention further relates to a method of typing genomic DNA which includes the step of forming a reaction mixture comprising the genomic DNA, an extension primer, a first amplification primer and a second amplification primer under conditions suitable to drive extension and amplification.
  • the extension primer comprises a known sequence and a first sequence ofgenomic DNAixonranother member of the species, the first amplification-primer specifically hybridizes to the known sequence and the second amphfication primer specifically hybridizes to a second sequence downstream from the first sequence of the genomic DNA of the other member of the species.
  • the presence of an extension product in the reaction mixture is then determined.
  • the invention also relates to a DNA adaptor which may be used in the preparation of extension primers according to the invention.
  • the adaptor according to the invention is a single stranded oligonucleotide capable of forming a partially double stranded structure having a closed end and an open end, the structure comprising at the closed end a first double stranded region, at the open end, a second double stranded region, between the first and second double stranded regions, a region of non-complementary sequences.
  • the non- complementary sequences comprise a known sequence to which a primer can specifically hybridize, and the second double stranded region comprises a class LTS RE recognition site.
  • the adaptor of the invention may be included in a kit which is also contemplated by the invention.
  • the invention also relates to primers generated according to the invention, including primers of SEQ ID NO. 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO. 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ED NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27
  • Figure 1(a) and 1(b) illustrate the structures of a Sequence-Capture (SC) Adaptor and an amplification adaptor, respectively.
  • the oligonucleotides that have their 5' ends phosphorylated were shown with a p at their ends.
  • Figure 2 is a schematic representation of amplification of unique DNA fragments by
  • Pigure_3 is. a ⁇ sehematic representation of amplification and reversal of die functional directionality of A-T-P, in accordance with an exemplary embodiment of the present invention.
  • Figure 4 is a schematic representation of amplification of DNA fragments from an in vitro bacterial genomic library using an anchor primer, in accordance with an exemplary embodiment of the invention.
  • Figure 5 is a schematic representation of preparation of A-T-P from an .amplified product of Figure 4, in accordance with an exemplary embodiment of the invention.
  • Figure 6 illustrates a portion of known map of E. coli dnak gene, illustrating the positions of the Pst I restriction sites and the PCR primer binding sites. Figure 6 also illustrates the amplified products obtained from extension/PCR reactions with A-T-PDNAK, PSS2 and 3 'DNAK, and with "reversed" A-T-PDNAK, PSS4 and 5'DNAK2.
  • Figure 7(a) is a summary of the PCR Products obtained from in vitro libraries (Pagl and EcoRI) of S. aureus strains with respect to known map of mec DNA regions.
  • Figure 7(b) is a summary of the PCR Products obtained from in vitro libraries (HindlH and Pstl) of S. aureus strains with respect to known map of mec DNA regions.
  • Figure 8 is a summary of the PCR Products obtained from in vitro library (EcoRI) of S. aureus strains with respect to known map of pSKl DNA.
  • Figure 9 is a summary of the PCR Products obtained from in vitro libraries (Pagl,
  • class IIS restriction endonuclease is used to describe a subgroup of class II RE that cleaves DNA at precise distances outside their recognition sites. Unlike class II RE that cut within the recognition site, class HS RE cuts DNA but leaves the recognition site intact. Depending on the individual class HS RE, the precise distance between the cleavage site and the recognition site, and the type of ends generated vary.
  • primer is used to describe single-stranded (ss) oligonucleotide that can be used to initiate DNA synthesis after hybridizing to its complementary DNA sequence in the presence of DNA polymerase and dNTP. It can also be used together with another primer in PCR reactions to amplify DNA fragments.
  • extension is used to describe the process of DNA synthesis initiated by an oligonucleotide primer.
  • Primer with "sufficient" complementary sequence to a DNA strand at its 3 ' end can hybridize to the DNA strand and has its length extended along the complementary DNA strand in the presence of DNA polymerase and dNTP.
  • “Sufficient” is used to mean the primer is thermodynarnically stable to remain bound to the DNA template strand, such that the DNA polymerase is able to extend the primer by copying the template strand.
  • sequence-capturing (SC) adaptor is used to describe an oligonucleotide that has complementary sequences to allow the formation of a partially double stranded structure which folds itself into a stem-loop structure as exemplified in Figure 1(a).
  • the open ends of these adaptors can be blunt, with a 5' overhang, or with a 3' overhang.
  • a unique sequence is present for PCR amplification.
  • the unique sequence is of a length sufficient for specific hybridization by a primer.
  • the uniqueness of this sequence is defined by the statistical probability of occurrence of a random sequence of that particular length.
  • This unique sequence may or may not contain a specific sites recognized by a restriction endonuclease.
  • One or more restriction sites recognized by a class HS restriction endonuclease is present in the adaptor.
  • captured sequence is used to describe the DNA sequence that is joined to the SC adaptor following ligation of the SC adaptor to a DNA fragment and digestion with a class IIS restriction endonuclease.
  • the length of the captured sequence is dependent on the class IIS restriction endonuclease used to cut the adaptor from the DNA fragment.
  • A-T-P adaptor-turned-primer
  • A-T-P The end of the A-T-P that is separated from the DNA fragment can be blunt, sticky end with 5' overhang, or sticky end with 3' overhang. A sticky end will facilitate ligation to an "amplification adaptor" described below.
  • A-T-P can be used as primer to initiate DNA synthesis.
  • A-T-P The term "functional directionality" of A-T-P is used to describe the extension direction of the A-T-P.
  • the direction of the synthesis is from the 5' end to the 3' end.
  • A-T-P When a primer hybridizes to the DNA template, only its 3' end can be extended.
  • A-T-P for extension, only the 3' end of the upper strand of the captured sequence can be extended.
  • the opposite strand of the captured sequence in the A- T-P is incapable of initiating primer extension. Therefore, only one strand of the A-T-P entity is functional as an extension primer and it determines the direction of the extension along the DNA template.
  • amplification adaptor is used to describe an entity formed from 2 oligonucleotides that have significant complementary sequences to allow the formation of a double-stranded (ds) region as exemplified in Figure 1(b).
  • One end of the adaptor is blunt.
  • a unique sequence is present for PCR amplification. This sequence has a length sufficient for specific hybridization by a primer.
  • This unique sequence may or may not contain specific sites recognized by a restriction endonuclease.
  • a restriction site recognized by a class HS restriction endonuclease is present.
  • the class IIS restriction endonuclease preferably has the same cutting pattern (i.e.
  • amplification adaptor cuts at the same distance away from the recognition site) as the class HS restriction endonuclease used to cut the SC adaptor.
  • This end of the amplification adaptor can be blunt, with a 5' overhang or with a 3' overhand.
  • a sticky end of a random sequence is a 2-bases 3' overhang and represents 16 (4 2 ) possible combinations of ends.
  • the amplification adaptor is used to reproduce A-T-P or to reverse the functional directionality of A-T-P.
  • in vitro library is used to describe genomic DNA that has been digested with an RE, and then ligated to SC adaptors. Genomic DNA is digested with different REs and then ligated to SC adaptors to-generate different in vitro libraries.
  • the present invention provides a method, referred to generally as SCOT (sequence capturing oligonucleotide technique) for preparing extension primers from unknown DNA sequences.
  • SCOT sequence capturing oligonucleotide technique
  • the extension primer can be used in an extension/amplification reaction to specifically extend the captured sequence and amplify the upstream sequences to the known 3 ' sequence or region.
  • the upstream sequence so amplified may then be sequenced or subject to other genomic analysis.
  • the extension/amplification reaction does not require prior sequence information of the captured sequence. This is in contrast to U.S patent no. 5,695,937 which discloses the use of a linker containing a class HS RE site to capture expressed sequence and formation of concatermers of the captured sequence.
  • the captured sequence then must be cloned and sequenced to provide quantitative and qualitative data about gene expression.
  • the extension primer of the invention may be amplified, and its functional directionality may be reversed.
  • extension primer generated by the present invention has a particular application in identification of polymorphic regions and therefore, to genotyping.
  • these extension primers can identity regions of polymorphism and thereby serve as genetic markers and/or provide the necessary sequence information to design such markers. While this aspect of the invention is exemplified below with respect to bacterial DNA strain typing, it will be readily appreciated that the invention extends equally to less complex genome and to other genome whose complexity is of the order of bacterial genome, including certain human cDNA or library clones of human DNA.
  • present-invention- may be -carried -in accordance -with- the teachings- that- follow- using standard techniques of molecular biology well known to persons skilled in the art.
  • Sequence Capturing is accomplished by ligating a SC adaptor to a DNA fragment with complementary sticky ends or with blunt ends. It is based on the cleavage principle of class IIS RE that cuts away from its DNA recognition site. Since the SC adaptor includes a class IIS restriction site, the class IIS RE is used to cut DNA ligated to the SC adaptor. The released SC adaptor now "captures" some DNA sequence from the DNA fragment that it ligated to. The number of bp captured is dependent on the class HS RE used; it generally ranges from 1 to 16 bp with variable sizes of overhangs. The class IIS RE recognition site can be positioned at the open end of the adaptor so as to maximize the length of the captured sequence.
  • the captured sequence should be of a length that enables sequence specific primer extension. That length will depend on the complexity of the DNA that is subject to analysis. The more complex the DNA, the more captured sequence is required. Preferably the length of the captured sequence is at least 12 bp long. More preferably, the captured sequence is 16 bp long. This length of captured sequence is sufficient to serve as a sequence-specific extension primer for human genomic DNA.
  • SC adaptor with a 4-bases 3' overhang is ligated to a
  • the ligation reaction may be purified for example with a DNA extraction kit (Fermentas, Lithuania).
  • the DNA extraction kit allows for the recovery of long DNA fragments by reversible binding of DNArto glass particles.
  • the iinligated adaptors are ⁇ removed-in-this ⁇ process .
  • the purified- fragment/adaptor is then digested with a class HS RE which recognizes the class HS RE site present in the SC adaptor.
  • the released SC adaptor contains some DNA sequence from the DNA fragment at its 3' end with respect to the top strand, and at its 5'end with respect to the bottom strand.
  • the A-T-P that results may be amplified, or it may be reversed with respect to its functional directionality, before its use in an extension/PCR reaction.
  • the DNA fragment used in the ligation could be a gel-purified fragment from a RE digestion, a DNA product of a PCR reaction, RE-digested plasmid, or RE-digested genomic DNA fragments.
  • the DNA may or may not be dephosphorylated depending on the SC adaptor to be used for ligation. If multiple DNA fragments are used in the ligation, the A-T-P generated will contain different captured sequences.
  • This A-T-P mixture can be used in an extension/PCR reaction but the number of different A-T-P' s (i.e. different captured sequences) should be limited so as to generate unique amplification products following extension PCR reaction. Preferably the number of different A-T-P' s is less than 20.
  • DNA fragments of high complexity bacterial genomic DNA
  • a specific DNA fragment from the ligation mixture is amplified by PCR as discussed below.
  • the PCR product may then be purified using agarose gel electrophoresis before subjecting to the class HS RE digestion to generate A-T-P.
  • A-T-P can be partially purified, for example, with molecular weight cut-off filters.
  • molecular weight cut-off filters By passing the class IIS RE digestion of fragment/adaptor through a 100,000 MW cut-off filter in a spin unit (Gilson), 80-90% of A-T-P can be separated from long DNA fragments and recovered.
  • Amplification of A-T-P ATP can be reproduced in large quantities by ligating to an amplification adaptor and amplifying the ligated product.
  • A-T-P is amplified in 10 PCR cycles to prepare a stock solution using primers that are complemenary to the known unique sequences on the SC adaptor and on the amplification adaptor.
  • Subsequent working solution of A-T-P is prepared from a 20-cycles PCR amphfication of a small aliquot of the stock A- T-P solution using a biotinylated amplification adaptor primer and the SC adaptor primer.
  • These PCR reactions can be carried out under the standard temperature cycling profiles used by those skilled in the art.
  • the duration of the temperature cycles in the PCR reactions should be modified according to the size of the amplified product. As the rates of polymerization by DNA polymerases are known, the appropriate time required to obtain a full length amplified product can be determined by those skilled in the art.
  • the PCR product is digested with the class HS RE that recognizes the restriction site on the SC adaptor.
  • the A-T-P is separated from the amplification adpator using streptavidin-immobilized beads.
  • the amplified A-T-P is functional in sequence-specific primer extension.
  • the amplified A-T-P/adaptor complex is first bound to the streptavidin beads.
  • the A-T-P is then released from the beads by digesting with a class US RE that recognizes the restriction site on the SC adaptor.
  • the supernatant containing the A-T-P is separated from the beads after a brief centrifugation step.
  • Affinity Tags other than biotin on the amplification adaptor primer can be used to separate ATP from the amplification adaptor after class US RE digestion.
  • A-T-P can be used to amplify a unique DNA fragment in an extension/PCR reaction.
  • the reaction is preferably carried out as a 2 step extension/PCR reaction.
  • A-T-P is added to a DNA mixture (e.g. genomic DNA) and the reaction is heat denatured. After the temperature of the reaction is brought down to 37°C, the A-T-P hybridizes to its complementary genomic DNA sequence and its sequence is extended with the addition of a DNA polymerase in the presence of dNTP. The newly synthesized-DNA- sfrand ⁇ now has Jhe.
  • the SC reaction is then carried out with a primer complementary to the unique sequence of the SC adaptor, and another primer that is specific to a region 3' downstream from the captured sequence.
  • the extension reaction is carried out with Klenow fragment in a PCR buffer. The extension reaction is carried out at 37°C for 30 min. At the end of the extension reaction, PCR primers and Taq DNA polymerase are added and the reaction is subjected to temperature cycling as in a standard PCR reaction.
  • the extension reaction may also be carried out with Taq DNA polymerase.
  • the extension reaction is carried out at 37°C for 30 seconds and then at 72°C for 2 minutes.
  • the extension reaction may be repeated, preferably 5 times.
  • PCR primers are added and the reaction is subjected to the standard PCR reaction conditions.
  • a specific DNA fragment is obtained comprising the SC adaptor sequence at the 5' end of the fragment.
  • the length of this fragment is determined by the distance between the captured sequence on the A-T-P and the 3' PCR primer.
  • A-T-P can be regenerated from this fragment by cutting with the class HS RE. If the A-T-P is functionally reversed as described below, it can be used to primer extend in the opposite direction.
  • A-T-P generated is a double stranded (ds) DNA molecule with either a blunt end or a sticky end.
  • the captured sequence is at the 3' end with respect to the top strand and 5' end with respect to the bottom strand of the A-T-P. Since primer extension is only carried out in the 5' to 3' direction, only the top strand can be used as sequence-specific primer with respect to the captured sequence.
  • the class HS RE which recognizes the restriction site on the amphfication adaptor, upon cleavage s ⁇ ou!d " preserve ⁇ the lehgthT of The ' captured sequence at least to the exferit necessary to enable sequence specific extension.
  • it has the same cutting pattern as the class US RE used to cleave SC adaptor.
  • the A-T-P/ amplification adaptor complex can be amplified in a 10-cycles PCR reaction as described for the amplification of A-T-P.
  • Subsequent working solution for preparing reversed A-T-P may be prepared from a 20-cycles PCR amplification of a small aliquot of the stock A-T-P solution using a biotinylated SC adaptor primer and the amplification adaptor primer.
  • the amplification reaction is digested with the class US RE that recognize the RE site on the amplification adaptor.
  • the captured sequence is now transferred to the amplification adaptor from A-T-P.
  • the bottom strand of the captured sequence is now at the 3' end of the amplification adaptor, and can be used in primer extension as sequence- specific primer. See Figure 3.
  • the amplified A-T-P/adaptor complex may be first bound to the streptavidin beads.
  • the reversed A-T-P is then released from the beads by digesting with a class HS RE that recognizes the restriction site on the amplification adaptor.
  • the supernatant containing the reversed A-T-P is separated from the beads after a brief centrifugation step.
  • the reversed A-T-P is functional in sequence-specific primer extension. Its functional (priming) direction is opposite to that of the A-T-P from which it was prepared.
  • PCR amplification of a DNA fragment requires two primers that are complementary to the sequence of opposite strands of the DNA fragment and that are some distance apart in their binding sites. Therefore, the DNA sequence information must be available in order to synthesize the primers. In the present invention, only one PCR primer complementary to a known DNA region is required to initiate the amplification process. In conjunction with an "in vitro library", specific DNA fragments around a known DNA region can be amplified with 1 target-specific primer hereinafter referred to as the "anchor primer”.
  • purified bacterial genomic DNA is digested with a restriction endonuclease. Following digestion, the genomic DNA may be purified. The digested DNA is then ligated to the SC adaptor. The ligation reaction may be subjected to PCR amplification without any further purification. See Figure 4.
  • the genomic DNA may be digested with any RE that generate cleaved fragments of a length that can be amplified.
  • the selection of the RE will depend on the DNA polymerase to be used to amplify the DNA fragment ligated to the SC adaptor.
  • multiple in vitro bacterial genomic libraries are created using restriction endonucleases that cut at a frequency of once per 2000 - 8000 bp in the bacterial genome. These restriction endonucleases produce 5' or 3' overhangs.
  • An anchor primer is required together with a primer specific to the adaptor (adaptor primer) to amplify DNA fragments from the in vitro genomic libraries.
  • a DNA fragment is amplified between a restriction site (the one used for creating the in vitro library) and the anchor sequence.
  • the anchor primer should specifically hybridize to variable positions within the bacterial genome to allow amplification of different DNA fragments from different regions of the bacterial genomes.
  • Candidates for the anchor sequence include insertion sequences and repetitive elements. The insertion sequences and repetitive elements are semi-conserved sequences that are present in multiple copies and in various locations of bacterial genomes.
  • an insertion element is chosen as the target for the anchor primer.
  • DNA fragments between the insertion element and the RE site is amplified. Polymorphism (between the insertion elements and the restriction sites in front of them) in terms of different lengths and/or absence of the amplified products may be observed among different bacterial strains. If more than one copy of the insertion element is present in the bacterial genome, then more than one band should be obtained unless the RE site is too Tar away from the1S ⁇ elerhent.
  • a PCR primer with sequence complementary to an internal region of the transposase gene in the IS431 insertion element was prepared as the anchor primer.
  • the ligation reactions are amplified with the adaptor primer and the anchor primer in PCR reactions. Any method of analysis to detect the amplification product and its size can be employed to analyze the amplified products. Agarose gel electrophoresis provides a convenient method for such analysis.
  • A-T-P The preparation steps of A-T-P are shown in Figure 5.
  • one or more bands not common to, and therefore capable of differentiating different strains or species is purified from a gel slice cut from the agarose gel.
  • the purified band is digested with the class IIS RE that recognizes the site on the adpator, to release the adaptor along with the captured sequence (A-T-P).
  • the A-T-P can then be ligated to an amplification adaptor.
  • the ligated A-T-P can then be ligated to an amplification adaptor.
  • P/adaptor complex is amplified in 10 cycles of PCR.
  • This PCR reaction can become the stock for that particular A-T-P and an aliquot of this stock PCR may be used to reproduce more A-T-P, with the adaptor primer and a biotinylated amplification adaptor primer, in 20 cycles of PCR.
  • the biotinylated product is then run in a 2.5% agarose gel or other appropriate concentration to separate it from any large contaminating fragments.
  • the short amplified product is cut from the gel and further purified with streptavidin beads.
  • the bound amplified products is digested with the class HS RE that recognizes the site on the adpator to release the A-T-P. The supernatant of the digestion is the working solution of A-T-P to be used in the extension/PCR reaction.
  • A-T-Ps are prepared from amplified products obtained from different in vitro libraries of different bacterial strains, or_species.. ⁇ As_discussed_above, each of the,ArT-P includes the adaptor primer sequence and the captured sequence from the original bacterial
  • A-T-Ps are used in extension/PCR reactions to amplify unique fragments from bacterial genomic DNA.
  • A-T-P is mixed with undigested bacterial genomic DNA of unidentified strain or species in the presence of Taq DNA polymerase. After denaturation, the upper strand of the A-T-P should be able to prime and extend on the genomic DNA if the complementary sequence of the captured sequence is present. After five repeat extension cycle, the reaction is mixed with the anchor primer and the adaptor primer. Exponential PCR is then allowed to proceed for 30 cycles. Amplified products would only be obtained if the captured sequence and the anchor sequence were located sufficiently close in the bacterial DNA.
  • the subject method therefore can be used to construct nucleic acid "fingerprints" of different bacteria strains.
  • fingerprints are specific to bacteria strains, and can be used to identify the type of bacteria in infections outbreaks.
  • A-T-Ps can be prepared from in vitro libraries of multiple strains of a particular species. Using the A-T-Ps in extension/PCR reactions of genomic DNA of the strains, a set of A-T-Ps that could distinguish all of the strains by the amplification products could be identified. By using this set of A-T-Ps, a unique DNA "fingerprints" will be obtained for each strain.
  • the amplified products of the extension/PCR reactions may also be sequenced.
  • Optimized PCR primers can then be designed around the captured sequence and are within the scope of the invention. They may be more effective than the original A-T-Ps in typing bacteria.
  • the extension/PCR reaction with A-T-P and 2 PCR primers in most cases is expected to be less sensitive than a PCR reaction using a pair of PCR primers. It may be useful to develop optimized PCR primers from amplified products for highly sensitive strain typing methods.
  • a database of PCR primers may be created to facilitate the development of a multiplex PCR approach to strain typing. Depending on the anchor primer used, the database could be applicable to a group of bacterial species.
  • the subject method therefore can also be used to identify and prepare DNA probes for a peulai - strain- or closely -related r-strains.
  • the amplified fragment can be made into a radioactive probe easily by including a radioactive nucleotide in the extension/PCR reaction.
  • modified primers are also within the scope of the invention.
  • This invention further contemplates a kit comprising one or more reagents of the invention.
  • the kit may include, in packaged form, a multicontainer unit having multiple SC adaptors of various sticky ends; an adaptor primer; an anchor primer; an amplification adaptor; an amplification adaptor, primer; each of four different nucleoside triphosphates; restriction endonucleases that produce ends corresponding to the sticky ends of the SC adaptors;. two class IIS restriction endonucleases that recognize restriction sites on the SC adaptor and the amplification adaptor; T4 DNA ligase; and an agent for polymerization of the nucleoside triphosphates. Instructions providing information to the user regarding the use of the kit can also be inserted, including for identifying polymorphic regions in bacterial genomic DNA.
  • the kit may also include biotinylated amplification and adaptor primers; and streptavidin beads for the purification of biotinylated products.
  • Example 1 demonstrates the sequence capturing function of the SC adaptor and the use of A-T-P as extension primer.
  • Example 2 illustrates the functional reversal of A-T-P by ligation to the reverser adaptor and PCR amplification.
  • Example 3 shows the creation of in vitro bacterial libraries and the preparation of strain-specific A-T-Ps. It also demonstrates the utility of A-T-Ps in the identification of polymorphic regions.
  • Example 4 illustrates the use of A-T-Ps in bacterial strain typing.
  • the sequences of the PCR primers are always in the same 5' to 3' direction as the non-complementary sequence of the SC adaptor.
  • Restriction endonuclease recognition sites are present in the SC adaptor. Kpn2I and Eco31I were incorporated for diagnostic purposes. Sail, Cfr42I, Eco52I and Notl are present for cloning the PCR products if required.
  • Gsul is the class HS restriction site for capturing sequence adjacent to the adaptor.
  • the open end of the adaptor has a sticky end that is complementary to the sticky end of a particular restriction endonuclease of interest. When ligated to a restriction digested DNA end, the restriction endonuclease recognition site is reconstituted.
  • Four SC adaptors with sticky ends corresponding to Pstl, EcoRI, Hindm and Pagl were used in our experiments for creating the in vitro libraries.
  • the SC adaptor oligonucleotide and PCR primers were chemically synthesized by MOBIX (McMaster University, Hamilton, Canada). Before using the SC adaptor for ligation to DNA fragments, the oligonucleotide was heated at 85°C for 5 minutes at a concentration of 25 pmol/ ⁇ l. The oligonucleotide was then allowed to cool slowly down to room temperature. These denaturation/renaturation steps allowed the oligonucleotide to fold into the desired "bubble" shape. The PCR primers were diluted to a working concentration of 25 pmol/ ⁇ l in ddH ⁇ O. The sequences of the adaptors and the primers are listed in Table 1.
  • E. coli genomic DNA was isolated from 10 ml of overnight DH5 ⁇ culture following the manufacmrer's protocol (Qiagen). The final concentration of the purified genomic DNA was 655 ⁇ g/ml. An aliquot of the purified genomic DNA (— 10 ng) was digested with 50 U of Pst I in IX O buffer (Fermentas) at 37°C for 1 hour. The digested genomic DNA was purified with phenol/chloroform mixture and ethanol precipitated. The DNA pellet was resuspended in 20 ⁇ l of ddH- . The final DNA concentration was estimated to be 288 fmol ends/ ⁇ l. Table 2. Pstl digestion of E. coli genomic DNA
  • a 20 bp oligonucleotide was synthesized corresponding to the E. coli dnaK gene as documented in the GenBank.
  • the primer sequence (3 'DNAK) is shown in Table 1. This primer was used in a PCR reaction with the SC adaptor primer (PSS2) to amplify a portion of the dnaK gene from the in vitro Pst I genomic library created in step 3 aoove.
  • the amplified DNA fragment would correspond to the part of the dnaK gene from the closest Pst I site to the location of the DnaK primer sequence (See Figure 6).
  • the expected size of the amplified product should be 375 bp long.
  • another oligonucleotide (5 'DNAK) corresponding to a 20 bp sequence in front of the
  • 3'DNAK primer was also synthesized (Table 1). When the 5' DNAK and 3'DNAK primers were used in a PCR reaction to amplify the portion of the dnaK gene from the in vitro Pst I genomic library, an amplified product of 290 bp long would be obtained.
  • the PCR reaction was set up as shown in the Table 4 below.
  • the PCR amplification was carried out in a Perkin Elmer thermocycler under the following conditions: 94°C for 3 minutes; 30 cycles of 94°C for 30 seconds, 65°C for 15 seconds and 72°C for 1 minute;
  • A-T-P preparation a) Purification and digestion of the amplified product
  • the sample PCR reaction (40 ⁇ l) was run on a 1% agarose gel as described before.
  • the amplified product was cut out from the gel and purified with the DNA Extraction kit (Fermentas).
  • the purified product was estimated to have a concentration of 10 pmol/ ⁇ l.
  • the purified DNA fragment was digested with Gsu I in IX B + buffer (Fermentas) at 30°C overnight to release the SC adaptor with the "captured sequence".
  • the digestion reaction was inactivated at 65 °C for 20 minutes. Double-distilled water was added to make the final volume to 100 ⁇ l.
  • the diluted reaction was spun through a Nanosep (Gelman) 100K MW cut-off filter unit at 5000 rpm in a microcentrifuge for 20 minutes. This step served to separate most of the long PCR product and the Gsu I restriction endonuclease from the A-T-PDNAK (the SC adaptor with the capmred sequence).
  • the filtrate containing the A-T-PDNAK was recovered.
  • the final concentration of the SC adaptor was estimated to be 2 fmol/ ⁇ l.
  • PCR was carried out using the SC adaptor primer, PSS2 and the amplification adpator primer, PSS4, in a reaction as described in the Table 7.
  • the PCR conditions was as follows: 94°C for 30 seconds; 10 cycles of 94°C for 5 seconds, 58°C for 5 seconds, and 72°C for 10 seconds; 72°C for 1 minute.
  • this reaction served as the stock solution for A-T-PDNAK.
  • a second round of PCR amplification was carried out with an aliquot of the first-round PCR reaction (Table 8).
  • the same PCR primer pair was used except that the PSS4 primer was biotinylated.
  • the same PCR ' conditions as T the first-round PCR were used except the ⁇ ⁇ al " numbef of cycle T was increased to 20.
  • a 10 ⁇ l aliquot of the second-round PCR reaction was ran on a 20% polyacrylamide gel in TBE buffer atl20V for 90 minutes.
  • second-round PCR reactions (200 ⁇ l) was ran on a 2.5 % agarose gel.
  • the biotinylated PCR product of 76 bp was cut from the gel.
  • the agarose slice containing the PCR product was spun in a spin- filter unit at lOOOxg for 10 minutes.
  • the biotinylated PCR product went through the filter with the liquid and was collected.
  • the biotinylated PCR product was ethanol precipitated and resuspended in 100 ⁇ l of ddELO.
  • the concentration of the biotinylated PCR product was estimated to be about 80 fmol/ ⁇ l.
  • Streptavidin beads (Dynal) were washed 2 times with 150 ⁇ l of 6X SSC buffer (0.9M NaCI, 0.09M Sodium citrate, pH 7.0), and then 2 times with 150 ⁇ l of 2X B&W buffer (lOmM Tris-HCl (pH7.5), ImM EDTA and 2M NaCI). After removing the wash liquid, the beads were resuspended in 50 ⁇ l of 2X B&W buffer. Fifty ⁇ l of the biotinylated PCR product was added to the Streptavidin beads in a microcentrifuge tube. The tube was taped to a rotating stage for 30 minutes at room temperature to ensure constant mixing.
  • the beads were washed 3 times with 150 ⁇ l of 2XJB&W ⁇ uffer,--and..then .3- times withulOO - ⁇ Lof X B- buffer. - The- wash -buffer was completely removed from the tube after the last wash.
  • the purified A-T-PDNAK was used to amplify a part of the dnak gene from E. coli genomic DNA in a novel extension/PCR reaction.
  • the first part of the reaction was the primer extension of the A-T-PDNAK on the genomic DNA using Taq DNA polymerase.
  • the capmred dnak gene sequence of the A-T-PDNAK would find its complementary sequence on the genomic
  • biotinylated PSS2 primer was used instead of PSS4 for the second-round PCR in step 5(c) of Example 1.
  • the complex was digested with the Type US restriction endonuclease which recognize the restriction site on the amplification adaptor. The captured sequence was released together with the amplification adpator from the beads.
  • This complex is termed the "reversed" A-T-P. It is functional in primer extension in the opposite direction as compared to the original A-T-P.
  • the beads with bound A-T-P/amplification adaptor complex were digested with Eco 571 that recognized the restriction site on the amplification adaptor (Table 13). After digestion at 37°C for 2 hours, the captured sequence was released together with the amplification adaptor. This molecule may now be used as "reversed" A-T-P.
  • Primer extension was carried out with the reversed A-T-PDNAK on E. coli genomic DNA as described in the Table 14.
  • the reaction conditions were the same as in step 6 of Example 1.
  • Example 3 Creation of in vitro bacterial genomic libraries and preparation of strain- specific A-T-Ps for S. aureus
  • Staphylococcus aureus is one of the major causes of noscomial infections and outbreaks in hospitals. Due to the epidemiological sigmficance of S. aureus, many typing methods have been developed and standardized using this organism. Therefore, S. aureus was used as a model in this example.
  • Genomic DNAs were purified from 10 ml of overnight cultures of these bacterial strains. Pelleted cells were washed and resuspended in 1 ml of TE buffer (pH 7.5). A 200 ⁇ l aliquot of each cell suspensions was mixed with 20 ⁇ l of Lysostaphin (0.01 ⁇ g/ ⁇ ) and 50 ⁇ l of lysozyme (50 mg/ml). The mixtures were incubated at 37°C for 1 hour to break up the cells. After the incubation period, 30 ⁇ l of 10% SDS and 50 ⁇ l of 7.5 M Sodium acetate were added to each mixture. The mixtures were phenol/chloroform extracted. The purified genomic DNA
  • DNAs were ethanol precipitated and resuspended in 100 ⁇ l.
  • the final concentrations of the genomic DNA from the 4 strains were between 0.4 - 1 ⁇ g/ ⁇ l.
  • IS element was chosen as the anchor sequence in our search for polymorphic regions in S. aureus.
  • IS257 element was found in direct repeats flanking the trimethoprim-resistance determinant on many members of the pSKl family of multi-resistance plasmids in S. aureus. It also forms direct repeats bounding mercury-resistance determinants on heavy-metal resistance plasmids; these repeats are also known as IS431 (which is an isoform of IS257 in the IS6 family).
  • IS257 sequences are found adjacent to chromosomal determinants for resistance to mercury, methicillin and tetracycline. Since the bacteria strains obtained from Nffl are all methicillin resistance, IS257 seemed like a good candidate.
  • a PCR primer with sequence complementary to an internal region of the transposase gene in IS431 insertion element was prepared.
  • the priming dhection of the primer was in the opposite direction of the open reading frame of the insertion element.
  • the sequence information of the insertion sequence was obtained from the IS431mec gene associated with methicillin resistance in 5". aureus (GenBank accession # X53818).
  • the anchor primer
  • a 5' primer (5' IS431) was also designed which is primarily used to provide a positive test for the presence of IS431 transposase sequence.
  • the uncut genomic DNAs were tested for the presence of the IS431 element using the ⁇ 5TS431 and 3 TS43F primers in PCR reactions " . " The corresponding 368 bp fragment " of the transposases gene was obtained for all of the four strains tested.
  • the ligation reactions described above may be used without the need for further purification.
  • the ligation products were amplified with the adaptor primer and the anchor primer (3'IS431) in PCR reactions as described in Table 24.
  • As a negative control S. aureus undigested and unligated genome DNA was used in place of the in vitro library in one of the PCR reaction.
  • As a positive PCR control 5TS431 and 3TS431 primers were used to amplify a fragment of the IS431 transposase gene from S. aureus genomic DNA.
  • the PCR reaction conditions were: 94°C for 2 minutes; 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 5 minutes; and 72°C for 5 minutes. Ten ⁇ l aliquots of each reaction were run on a 1 % agarose gel.
  • amplified products of different lengths were obtained from different libraries for each strain.
  • the lengths of the amplification products for a particular library were sometimes the same between two strains but never the same for all four strains.
  • the exception was the HindHI libraries. This was due to the presence of a Hindm site just 200 bp in front of the 3TS431 primer.
  • 3OEGIS431 (DE) primer was used as the anchor primer in the PCR reactions of the Hmdi ⁇ libraries, the lengths of the amplified products were again different among the strains; Strain 1 and Strain 4 shared a common 1900 bp band, and Strain 2 and Strain 3 shared a common 800 bp band.
  • the right side of the Table 24 shows the results obtained when the 3' degenerate primer was used as the anchor primer. From documented sequence data, the positions of the SS primer and the DE primer are about 250 bp apart. In all cases, the amplified bands obtained from PCR reactions using the DE primer were judged to be fairly similar in size to those obtained from the SS primer and we adopted a general practice of labelling the length of the amplified products obtained from DE primer as 250 bp smaller than the corresponding bands obtained from SS primer.
  • the 1100 bp difference between the two different product lengths was due to the different insertion sites of the IS431 element in different strains.
  • the 3' end sequence homology for all of the strains represent the common sequence ( ⁇ 470 bp) of the IS431 insertion element.
  • A-T-P was prepared from the amplified products obtained above.
  • the preparation of A-T-P was carried out as described in step 5 of Example 1 and shown in Figure 5. Thirteen A-T-Ps were prepared from the amplification products of the in vitro libraries of the 4 S. aureus strains.
  • One of the 13 A-T-Ps (obtained from Strain 1) only produced an amplification product with the genomic DNA from which it was generated ⁇
  • This mdicated__the strictlycaptured_sequence- is -unique -to -Strain A; that is,- the- captured sequence is not present in other strains, or that the anchor primer sequence in other strains is not close enough to the captured sequence for PCR amplification to occur.
  • at least nine out of the thirteen A-T-P prepared were informative and functional in extension/PCR. This represents a 70% success rate in .the preparation of informative and functional A-T-P
  • Extension/PCR reactions were carried out with uncut S. aureus genomic DNA from the 4 strains using the 9 functional A-T-Ps. Extension reactions with different A-T-Ps were set up as described in the Table 26 and heated at 94°C for 2 minutes. The extension reactions were repeated 5 times under the following temperature profiles in a thermocycler: Thirty seconds at 94°C, 30 seconds at 37°C and 5 minutes at 72°C.
  • the PSS2 primer and the 3TS431 or the 3'DEGIS431 primer were added to each extension reaction.
  • the second step of the extension/PCR reaction (Table 27) was carried out under the foUowing conditions: 94°C for 2 minutes; 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes; 72 minutes for 5 minutes. Table 27. PCR amplification of the extension reaction
  • Results are shown in Table 28.
  • A-T-P13 and A-T-P14 were redundant as they were prepared from the same restriction cut library (HindTII) but from different strains (2 and 1, respectively).
  • the lengths of the amplified products from extension/PCR reactions were the same further indicated that they were redundant; that is, they have the same captured sequence. This condition also applied for A-T-P10 and A-T-Pll, and A-T-P8 and A-T-P3.
  • the patterns of amplification products obtained from extension/PCR for the four strains can be classified into 4 groups.
  • Group 1 pattern obtained from A-T-P2, A-T-P7, A-T-P13, and A-T-P14, showed that Strain 1 and Strain 4 have common amplified products, and Strain 2 and Strain 3 have another common amplified products.
  • the PCR products obtained from the in vitro Pstl libraries were sequenced, they were found to correspond to the mec DNA region. Therefore, the A-T-Ps prepared from them should amplify fragments from the mec DNA region. From the GenBank sequence of mec DNA, the Pagl and Hindm sites can be determined so that the correct sizes of the extension/PCR products from the corresponding A-T Ps_ can_be_predicted.
  • A-T-P-7 was 100 bp smaUer than that of A-T-P-2, and the amplification products of A-T-P13 and A-T-P14 were 200 bp longer than that of A-T-P2.
  • Group 2 pattern obtained from A-T-P 6, showed that only Strain 2 and Strain 3 have amplified products of the same length.
  • the absence of amplified products for Strain 1 and Strain 4 could mean that the captured sequence is not present in these strains, or the
  • IS431 element is not inserted close by the captured sequence and/or in the correct orientation.
  • Group 3 pattern obtained from A-T-P10 and A-T-P 11, showed that only Strain 3 and Strain 4 have amplified products of the same length.
  • Group 4 pattern obtained from A-T-P8 and A-T-P 3, showed that only Strain 1 and Strain 3 have amplified products of the same length.
  • Group 1 pattern represents the mec DNA region as discussed earlier.
  • Group 2 pattern turned out to represent a second known IS431 insertion site further downstream from the first IS431 site on the mec DNA region. It is interesting to note that only Strain 2 and Strain 3 have this second insertion site.
  • Group 3 pattern was mapped to a Staphylococcus multi-resistance plasmid DNA (pSKl) known to be associated with trimethoprium resistance and has 3 copies of IS257 elements (GenBank Accession #X13290). Only Strain 3 and Strain 4 have this plasmid sequence.
  • Group 4 pattern was mapped to a Staphylococcus conjugative plasmid DNA (pSK41) known to have multiple copies of IS257 elements.
  • Figures 7-9 summarize the locations of the PCR products (in Table 25) obtained from the different libraries and the different strains with respect to the four DNA regions mapped. The locations were deduced from A-T-P extension/PCR experiments, and sequencing data obtained from PCR products produced from the libraries. PCR products obtained with the specific 3TS431 anchor primer and the degenerate 3'DegIS431 anchor primer are indicated as SS and DE, respectively. The PCR products from which A-T-Ps were prepared are indicated with broad arrows in these Figures.
  • Figure 7 shows the mec DNA region with the 2 copies of IS431 elements as documented in the GenBank sequence.
  • Figure 7(a) the PCR products obtained with Pagl and EcoRI libraries are shown.
  • AU 4 strains have the first IS431 element albeit the IS431 elements of Strain 2 and Strain 3 are inserted closer to the Pagl site.
  • There is no EcoRI site close by the first IS431 element so PCR products for EcoRI in vitro libraries were not obtained for this region.
  • SEQ ID NOS: 22 to 27 represent the sequence of the A-T-P including the capmred sequence of SEQ ID NOS: 16 to 21, respectively.
  • Figure- 9 the--pSK41 -plasmid- -DNA -containing 7 copies- of- IS257- elements -is- shown. There are no Pstl sites in the pSK41 DNA.
  • Figure 9(a) shows the left-halves of the pSK41 in close-up.
  • A-T-P S3PagI8 prepared from the PCR product from the Pagl library of Strain 3 was instrumental in the identification of this plasmid DNA in extension/PCR reactions. Only Strain 1 and Strain 3 produced 800 bp products in extension/PCR reactions (Table 28). The DNA sequence of the 800 bp PCR product matched the region in front of the first IS257 element in pSK41 DNA.
  • PCR products of —860 bp should also be obtained from Pagl libraries of Strain 1 and Strain 3. These bands would be so close to the 800 bp products that we would not be able to separate them on agarose gels used in the experiments.
  • the 800 bp PCR products from Strain 1 and Strain 3 (Pagl libraries, Table 25) were determined to be comprised of multiple sequences by running the sample in higher percentage agarose gels (before A-T-P was prepared). From sequencing the "non-purified" 800 bp product of Strain 1, it was found that the sequencing data was not readable suggesting that the product comprised of multiple sequences.
  • A-T-P (SlEcoRI40) was prepared from this PCR product. When this A-T-P was used in extension/PCR reactions, only the genomic DNA of Strain 1 produced a similar sized band.
  • This DNA product was not sequenced, but the size (4000 bp) matched what would be expected if it comes from the region in front of the third IS257 element of pSK41 DNA.
  • Figure 9(b) shows the right-halves of pSK41 DNA.
  • the 2500 bp PCR product obtained from the Hindm library of Strain 1 was tentatively assigned to this region.
  • the product size matches the region in front of the fifth IS257 element of pSK41 DNA.
  • Table 30 summarizes the information obtained from these experiments on the four strains of S. aureus. Basically, these 4 S. aureus strains can be distinguished using only 4 A-T-Ps (that is, four captured sequences). As the sequences of the four variable regions have-been determined, optimized-PGR- primers for multiplex PCR can -be prepared -and the strains can be distinguished using multiplex PCR with the four optimized PCR primers. Example 4. Use of A-T-Ps in bacterial strain typing
  • the results obtained with the 3TS431 (SS) anchor primer and 3OEGIS431 (DE) anchor primer are shown in Table 31 and Table 32, respectively.
  • the 16 strains of S. aureus can be assigned into 11 groups. With the exception of Group E (6 members), every group only has 1 member. For Group E, the mec DNA regionl was the only DNA region detected by extension/PCR reactions. Therefore, new additional A-T-P would be needed to further sub- classify these strains; additional A-T-P could be prepared from in vitro libraries of this group.
  • anchor primer was used in the extension/PCR reactions with A-T-P( S2PAGI20). At this time, it is not known what is the exact reason that additional bands were obtained for these strains.
  • One possibility is that the transposase gene sequences of the insertion elements in these strains are slightly different than the sequence of the IS431 anchor primer. Therefore, only the degenerate anchor primer (3OEGIS431) can hybridize to the DNA of these strains to give amplified products.
  • the screening result was not that different from the result obtained with the SS anchor primer.
  • the strains screened can still be classified into 11 groups. Five members (Strains 5, 7, 8, 10, and 14) of the previously classified Group E are now re-classified to Group B. Strain 9 becomes the lone member in Group E. None of the members of the other groups changed.

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Abstract

La présente invention concerne un nouveau procédé d'analyse génomique. Selon ce procédé, une amorce d'extension, produite par capture d'une séquence d'ADN inconnue au moyen d'un adaptateur contenant un site de reconnaissance à endonucléase de restriction de classe IIS, est utilisée dans une réaction d'extension/d'amplification avec un échantillon d'ADN, afin d'amplifier un produit unique contenant la séquence capturée. Le procédé selon cette invention permet d'identifier et d'analyser des séquences inconnues situées en amont d'une région ou d'une séquence connue. Cette invention s'applique notamment au génotypage, tel que le typage de souches bactériennes. De manière spécifique, cette invention concerne un procédé pour préparer des amorces d'extension, afin de capturer des séquences à différents emplacements génomiques, ainsi que leur utilisation en tant que marqueurs génétiques. En outre, cette invention concerne des adaptateurs permettant de préparer des amorces d'extension.
PCT/CA2001/001487 2000-10-23 2001-10-22 Nouveau procede d'analyse genomique WO2002034939A2 (fr)

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