Classifications

• • 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
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/682—Signal amplification

Definitions

• the present invention relates generally to a method of amplifying closed circular nucleic acid probes and, more particularly, to a method of amplifying closed circular nucleic acid probes by rolling circle amplification.
• the method of the present invention is useful in a range of applications involving the detection of nucleic acid sequences such as, but not limited to, the identification of genetic disorders, genetic variants or the presence of microbiological or viral agents.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• TAS transcription-based amplification system
• Strand displacement amplification (SDA) (7) is an isothermal technique which relies on the ability of a restriction enzyme to nick a hemiphosphorothioated recognition site and the ability of a polymerase to initiate replication at a nick and displace the downstream strand.
• the other isothermal technique which can be used to amplify a nucleic acid sequence is rolling circle amplification (RCA).
• the circular probe commonly referred to as a "padlock probe”
• the circular probe is designed such that it has regions at both its 5 ' and 3 ' ends which are complementary to the target sequence of interest and are separated by a region of nucleotides of non-target derived origin.
• the 5' and 3' ends of the probe are brought into close proximity to one another. If the two probe regions are adjacent to one another the 5' and 3' ends can be joined to produce a circular probe. In some instances, however, the probe regions are separated from one another by a small stretch of nucleotides. This region must be filled to achieve the generation of a circular probe.
• a variety of techniques can be utilised including the use of spacer oligonucleotides or by using a DNA polymerase (or a reverse transcriptase in the case of an RNA target) in combination with deoxynucleotide triphosphate molecules to fill the gap prior to ligation.
• a significant problem associated with the rolling circle amplification technique is the occurrence of background amplification.
• this background amplification was dismissed as primer-induced deletion fragment repeats encompassing a full unit repeat minus the intervening region between 5 ' ends of the two primers (8).
• Background amplification represents both a significant problem and a limitation for rolling circle amplification reactions which utilise 2 primers. It is also a major source of false positive results.
• the magnitude of the problem presented by the occurrence of this background amplification has been such that it has not been feasible to use the two primer rolling circle amplification techniques with an acceptable level of specificity.
• AmpX This class of background amplification has been termed "AmpX".
• the inventors have determined that it is an alternative amplification reaction which utilizes any linear nucleic acid probe molecules present in the reaction mixture. Typically the reaction products are multimers of head to tail tandem repeats. However, the inventors have determined that rather than encompassing sequence from the entire circular probe, the products of the AmpX reaction include repeats of a region of the linear target molecule that includes the two primer binding sites, the intervening sequence and some additional sequence of the template molecule flanking the primer binding sites.
• the inventors have developed a method for minimizing AmpX background amplification by enriching for closed circular nucleic acid probe molecules prior to their amplification.
• the amplification step utilising an enriched population of closed circle nucleic acid probe molecules the incidence of background amplification caused by the AmpX reaction is significantly reduced, thereby enabling more specific rolling circle amplification to occur.
• nucleotide sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography.
• Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210 > followed by the sequence identifier (e.g. ⁇ 210> 1, ⁇ 210>2, etc).
• the length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by informatio provided in the numeric indicator fields ⁇ 211 > , ⁇ 212> and ⁇ 213 > , respectively.
• Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field ⁇ 400> followed by the sequence identifier (e.g. ⁇ 4001 > 1, ⁇ 400>2, etc).
• one aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
• Another aspect of the present invention provides a method of rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• Still another aspect of the present invention more particularly provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• a further aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enzymatically enriching for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
• Still a further aspect of the present invention provides a method of multiple primer rollin circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
• Yet another further aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising non-enzymatically enriching for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
• Still yet another further aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
• Yet another aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• Yet a further aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a biotinylated capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe.
• Yet another aspect of the present invention provides a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment.
• Still another aspect of the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment.
• Still yet another aspect of the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; and generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe.
• the improvement comprising amplifying a circular probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching for said circular nucleic acid probe and then subjecting said circular nucleic acid probe to amplification.
• the method of rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enrichin for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the improvement comprising the steps of facilitating the interaction of a nucleic acid pro with a target nucleic acid sequence; generating a circular nucleic acid probe and enrichin for said circular nucleic acid probe by enzymatic enrichment; and subjecting said enriche circular nucleic acid probe to amplification.
• the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment; a subjecting said enriched circular nucleic acid probe to amplification.
• the present invention provides in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of sa probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucle acid probe to amplification.
• Another aspect of the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating t interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecular wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nuclei acid probe, incorporating a capture ligand into the region intervening said terminal region and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• kits for facilitating rolling circle amplification comprising compartments adapted to contain any one or more of nucleic acid probes, enzymes, capture ligands, means for isolating circular nucleic acid probes and reagents useful for facilitating circularisation, isolation and amplification of said probes. Further compartments may also be included, for example, to receive biological samples.
• Figure 1 is a schematic representation of Rep mediated circle ligation.
• Figure 2 is a schematic representation of a nucleic acid probe incorporating a GCN4 recognition sequence ( ⁇ 400 > 26) .
• Figure 3 is a schematic representation of capture by differential hybridisation.
• Figure 4 is schematic representation of padlock FV2.
• ⁇ 400> 13 depicts the padlock FV- sequence and ⁇ 400> 14 the LigW spacer.
• ⁇ 400> 16 depicts the Wildtype target and ⁇ 400 > 24 and ⁇ 400 > 25 the amplification primers.
• Figure 5 is a photographic representation of isothermal amplification from a synthetic target molecule.
• Panel A is a diagrammatic illustration of the synthetic target molecule and primers used in the amplification reaction ( ⁇ 400 > 1- ⁇ 400 > 8). Various combinations of these primers were used in isothermal amplification reactions.
• Followin amplification 10 ⁇ L of products from each reaction were loaded onto a 2% w/v agarose gel alongside a 100 bp ladder (lane M), electrophoresed and visualized by ethidium bromide staining (Panel B).
• Reactions included a control using primers B4C/P5C to which no synthetic target was added (lane C), as well as reactions using primers P1/B4C (lane 1), P5C/B4C (lane 2), P5C/B4 (lane 3), P5C/B4I (lane 4), P4D/B4C (lane 5), P5D/B4I (lane 6) and P5E/B4I (lane 7).
• Figure 6 is a photographic representation of restriction digest and sequence analysis of amplification products.
• Products for four separate amplification reactions using primers B4C/P5C (Panel A), B4I/P5E (Panel B), B4I/P5D (Panel C) and B4C/P1 (Panel D) were analaysed by restriction digestion and sequence analysis.
• An aliquot of each reaction was digested separately with Sau3Al (lane S), AM (lane A), EcoRI (lane E) and Taql (lane T) and electrophoresed through 2% w/v agarose gel alongside a 20 bp ladder (lane M) and a undigested control (lane U).
• Figure 7 is a photographic representation of sensitivity of amplification reaction. Serial dilutions of the synthetic target molecule were made and used as templates for the amplification reactions. Following amplification 10 ⁇ L of product from each reaction were electrophoresed through 1% w/v agarose gel and visualized by ethidium bromide staining.
• Figure 8 is a photographic representation of the detection of mini-transposon containing E.coli.
• Primers designed to amplify a 120 bp region of the KanR mini-transposon ( ⁇ 400>9) are illustrated (Panel A), together with the oligonucleotide, In903 ( ⁇ 400 > 11), used as an internal hybridization probe.
• the primers IF ( ⁇ 400 > 10) and 1R ( ⁇ 400 > 12) were used in reactions to amplify the mini-transposon sequence from various amounts of E.coli PNG801 genomic DNA, carrying this particular mini- transposson.
• Amplification reactions were also carried out on various amounts of E.coli DH5cc genomic DNA, as a negative control following amplification 1 ⁇ L of product was digested with Hindlll (lane H) and electrophoresed through a 2% w/v agarose gel alongside an undigested control (lane U) and molecular weight markers (lane M). Contro reactions were also included to which no template was added.
• Figure 9 is a schematic diagram of padlock hybridisation and circularisation.
• Figure 10 is a photographic representation of RCA reactions on synthetic targets using either unpurified or purified templates. Duplicate sets of ligation reactions were setup at outlined on page 1 (tagged spacer protocols). The oligonucleotides used for this reaction are illustrated diagramatically (Fig. 11). Two separate tubes were included for each reaction set.
• the negative control reaction (-ve) contained Padlock FV2 and LigW while the positive control reaction (+ve) contained Padlock FV2, LigW and Wildtype target oligonucleotides.
• l ⁇ L of Ampligase was added to the positive control reactions only. Ligation reactions were carried out at 60 °C for 1 hour. One set of ligations was then purified by the described method.
• Figure 11 is a schematic representation of oligonucleotide design used for rolling circle amplification detection of the normal and mutant alleles of the Factor V Leiden gene detection Using RCA.
• Padlock FV2 ⁇ 400> 13; LigW Spacer: ⁇ 400> 14; LigM spacer: ⁇ 400> 15; Wildtype target: ⁇ 400> 16; Mutant target: ⁇ 400> 17; Primers: ⁇ 400> 18 and ⁇ 400> 19).
• Figure 12 is a photographic representation of the comparison of unpurified and purified templates for RCA reactions.
• Figure 13 is a photographic representation of the sensitivity of RCA from purified circles
• Ten fold serial dilutions of purified circular molecules were used as templates for RCA reactions at 60° C for 2.5 hours.
• the number of circles present in each reaction were estimated to be 8xl0 9 (lane 1), 8xl0 8 (lane 2), 8xl0 7 (lane 3), 8xl0 6 (lane 4), 8xl0 5 (lane 5), 8xl0 4 (lane 6), 8xl0 3 (lane 7), 8xl0 2 (lane 8), 8x10' (lane 9) 8 (lane 10), 0.8 (lane 11
• a negative control reaction was also included to ensure no primer artifacts we generated during the course of the reaction.
• Figure 14 is a graphical representation of real time visualisation of rolling circle amplification reactions.
• Figure 15 is a graphical representation of the linear relationship between number of circles present and time to reach threshold fluorescence.
• Figure 16 is a photographic representation of the ability of RCA to specifically detect a gene of interest.
• Figure 17 is a schematic representation of the oligonucleotide design for SNP detection of factor V Leiden normal and mutant sequences (Wildtype sequence: ⁇ 400>20; Mutant sequence: ⁇ 400>21; FV5: ⁇ 400 > 22; FV6: ⁇ 400 >23; FV3: ⁇ 400 > 18).
• Figure 18 is a photographic representation of the potential of RCA for SNP detection.
• Figure 19 is a schematic representation of a padlock probe designed such that no two primers bind to any one oligonucleotide.
• Figure 20 is a photographic representation of the solution phase material produced in the reaction described in Example 11.
• Figure 21 is a photographic representation of solution phase material produced in the reaction described in Example 12. DETAILED DESCRIPTION OF THE INVENTION
• the present invention is predicated, in part, on the identification of a class of non-specific background amplification reaction which occurs during the amplification of circular probes, such as during rolling circle amplification.
• This class of non-specific amplification is termed "AmpX" and has been identified by the inventors as occurring due to the presence, in the reaction mixture, of linear nucleic acid probes and open circle nucleic acid probes. Accordingly, the inventors have developed a method of minimising AmpX non-specific amplification by incorporating into the amplification protocol the step of enriching for the closed circular probe molecules prior to their amplification. This step may be achieved, for example, by the purification of closed circle molecules or the removal of linear and/or open circle molecules.
• one aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching for said circular nucleic acid probe and then subjecting said nucleic acid probe to amplification.
• the steps of generating a circular nucleic acid probe and the enriching for said probe may be performed in any order. That is, the hybridised probe may be circularised prior to its enrichment or enrichment for the hybridised nucleic acid probe may be performed prior to its circularisation. Further, any one or more steps of the method of the present invention may be performed sequentially or simultaneously.
• the present invention provides a method of rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• rolling circle amplification is not to be taken to refer to a particular form of amplification or a particular amplification protocol. It should be understood to refer to any method of amplifying a circular nucleic acid molecule.
• interaction should be understood as a reference to any form of interaction such as hybridisation between complementary nucleotide base pairs or some other form of interaction such as the formation of bonds between any nucleic acid or non-nucleic acid portion of the probe molecule with any nucleic acid or non-nucleic acid portion of the target molecule.
• the interaction may occur via the formation of bonds such as, but not limited to, covalent bonds, hydrogen bonds, van der Waals forces or any other mechanism of interaction.
• All references hereinafter to "hybridisation” between two nucleic acid molecules should be understood to encompass any form of interaction between said molecules, for example, where said molecules become associated due to the interaction of non-nucleic acid components of said molecules.
• nucleic acid probe should be understood as a reference to any molecule comprising a sequence of nucleotides, or functional derivatives thereof, the function of which includes the hybridisation of at least one region of said nucleotide sequence with a target nucleic acid sequence.
• target nucleic acid sequence is a reference to any molecule comprising a sequence of nucleotides or functional derivatives thereof which molecule is a molecule of interest and is therefore the subject of identification via a probing step. Both the nucleic acid probe and the target nucleic acid sequence may comprise non-nucleic acid components.
• the nucleic acid probe may also comprise a non-nucleic acid detection tag or some other non-nucleic acid component which facilitates the functioning of the molecule.
• the target nucleic acid sequence may comprise a non-nucleic acid component.
• the target nucleic acid sequence may be bound to an antibody. This may occur, for example, where the target nucleic acid sequence is present in a biological sample isolated from an individual who is mounting an immune response, such as an autoimmune response, to said target nucleic acid sequence.
• the nucleic acid probe may be a protein nucleic acid which comprises a peptide backbone exhibiting nucleic acid side chains.
• nucleic acid probe should also be understood to encompass reference to two or more nucleotide sequence molecules which are ligated, associated or otherwise joined such that they form a single nucleotide sequence molecule which ligation or other form of joining is performed either during or after probing of the target sequence with the nucleic acid probe. Accordingly, facilitation of the ligation or other form of association of the nucleotide sequence molecules may be performed at any time during or after probing of the target sequence such as before, during or after hybridisation of the nucleic acid probe to the target sequence. For example, in the Rep mediated system of ligation (an example of which is represented schematically in Figure 1), a target sequence is probed with two nucleic acid molecules.
• a first probe molecule comprises a terminal TATTATT sequence while a second probe molecule comprises a terminal TATTATTAC sequence.
• the Rep molecule is utilised to facilitate cleavage of the TATTATT component of the terminal TATTATTAC of said second probe molecule followed by ligation of the terminal AC of said second probe molecule to the terminal TATTATT component of a first probe molecule which has hybridised to the target sequence, for example, at a position adjacent to said second probe molecule.
• the nucleic acid probe is preferably a single stranded nucleotide sequence and may have any conformation including, for example, a linear conformation or an open circle confirmation, that is, where the nucleotide probe is substantially circular in shape but its terminal regions do not connect.
• Reference to the "terminal regions" of the nucleic acid probe is a reference to the region located at each end of the nucleic acid probe.
• the nucleic acid probe preferably comprises two discrete target probe regions located one at each terminal region of the nucleic acid probe. However, it should be understood that the target probe regions are not necessarily located at the terminal regions of the nucleic acid probe and may be located at any other suitable region of the nucleic acid probe.
• the target probe region is the region of nucleotides complementary to one or more nucleotide sequence regions of the target nucleic acid sequence of interest.
• the nucleotide sequence region located between the terminal regions of the nucleic acid probe also preferably comprises at least one primer region.
• the "primer region” is a reference to the sequence of nucleotides which are designed to interact with at least part of a primer. Reference to the "primer region” also encompasses reference to any sequence of nucleotides to which a sense primer corresponds.
• the primer is a molecule comprising a nucleotide sequence which interacts with a region of a target nucleic acid sequence and from which complementary nucleotide synthesis, for example utilising a polymerase such as DNA polymerase, is initiated.
• the interaction of a primer with a primer region may occur by any suitable means such as, but not limited to, hybridisation of complementary base pairs or the interaction of non-nucleic acid components comprising the primer and the primer region.
• the nucleic acid probe may also optionally comprise regions corresponding to replication of origins, promotors, nucleic acid and/or non-nucleic acid detection tags.
• the nucleic acid probe comprises two target probe regions and two primer regions wherein exponential amplification of the circular nucleic acid probe is achieved due to the interaction of a first primer with a primer region of the nucleic acid probe and a second primer which interacts with a region of the nucleic acid probe complementary strand which is synthesised following interaction of the first primer with the probe.
• This method of rolling circle amplification is herein referred to as "two primer rolling circle amplification" .
• the primers may function by any suitable means.
• the first primer may be designed to interact via complementary base pairing with a primer region of the nucleic acid probe.
• This type of primer is commonly referred to as a complementary primer and facilitates the synthesis of a nucleic acid strand complementary to the nucleic acid probe.
• the second primer may be designed as a sense primer which corresponds to a second primer region of the nucleic acid probe thereby facilitating the synthesis of a nucleic acid strand complementary to the strand synthesised utilising the first primer.
• a single primer nucleotide sequence may be used which primer recognises two or more distinct primer regions of the nucleic acid probe.
• the primer regions may be of complementary nucleotide sequence orientation.
• one or more of the primers may comprise a non-nucleic acid component which interacts with a nucleic acid or non- nucleic acid component at a primer region thereby facilitating the synthesis of a complementary nucleic acid strand.
• the method of the present invention extends to amplification which utilises more than two primers (referred to herein as “multiple primer amplification").
• Multiple primer amplification should be understood to include the use of a single sequence which recognises two or more distinct primer regions of a nucleic acid probe.
• the present invention more particularly provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the multiple primer rolling circular amplification is two primer rolling circle amplification.
• nucleic acid should be understood as a reference to both deoxy ribonucleic acid and ribonucleic acid or derivatives thereof.
• the nucleic acid molecules utilised in the method of the present invention may be of any origin including naturally occurring (for example a biological sample may be utilised), recombinantly produced or synthetically produced.
• a biological sample is utilised, for example as a potential source of target nucleic acid sequence
• the nucleic acid component may optionally be extracted from the sample prior to testing (for example for the purpose of coupling it to a solid phase such as paper). This is not essential, though, and the method of the present invention may be performed utilising, for example, blood samples or it may be performed in situ with a biopsy specimen.
• “derivatives” should be understood to include reference to fragments, parts, portions, chemical equivalents, analogues, mutants, homologous and mimetics from natural, synthetic or recombinant sources. "Functional derivatives” should be understood as derivatives which exhibit any one or more of the functional activities of nucleotides or nucleic acid sequences.
• the derivatives of said nucleotides or nucleic acid sequences include fragments having particular epitopes or parts of the nucleotide or nucleic acid sequence fused to other proteinaceous or non-proteinaceous molecules.
• Analogs contemplated herein include, but are not limited to, modifications to the nucleotide or nucleic acid sequence such as modifications to its chemical makeup or overall conformation.
• nucleotides or nucleic acid sequences interact with other nucleotides or nucleic acid sequences such as at the level of backbone formation or complementary base pair hybridisation.
• biotinylation of a nucleotide or nucleic acid sequence is an example of a "functional derivative" as herein defined.
• Derivatives of nucleic acid sequences may be derived from single or multiple nucleotide substitutions, deletions and/or additions.
• the term "functional derivatives" should also be understood to encompass nucleotides or nucleic acid sequences exhibiting any one or more of the functional activities of a nucleotide or nucleic acid sequence, such as for example, products obtained following natural product screening.
• Facilitating the interaction of the nucleic acid probe with the target nucleic acid sequence may be performed by any suitable method. Those methods will be known to those skilled in the art.
• the nucleic acid probe assumes an open circle conformation (herein referred to as an "open circle nucleic acid probe").
• the target probe regions By interacting with a target nucleic acid sequence, the target probe regions generally form two discrete duplex regions due, for example, to complementary nucleotide base pairing between the nucleotides of the target nucleic acid sequence and the nucleotides of the target probe region of the nucleic acid probe (referred to herein as "duplexes").
• duplexes exist non-contiguously due to the absence of a bond, such as the phosphodiester bond, between the terminal nucleotide at the 5' end of the nucleic acid probe and the terminal nucleotide at the 3' end of the nucleic acid probe.
• a bond such as the phosphodiester bond
• open circle nucleic acid probe should be understood to also encompass the formation of a single open circle configuration which comprise two or more nucleic acid probes.
• a double open circle nucleic acid probe (which is encompassed within the meaning of "open circle nucleic acid probe") is formed where:
• Said first and second target nucleic acid sequences may be identical or different.
• a double open circle nucleic acid probe therefore exhibits two pairs of non-contiguous duplexes. One is located on the first target nucleic acid sequence and one is located at the second target nucleic acid sequence.
• Open circle probes of this type operate similarly to the single probes and yield identical products.
• These multiple open circle nucleic acid probes are circularised and enriched for in the same manner as open circle probes comprising only a single probe. In fact, any given reaction mixture is likely to comprise open circle nucleic acid probes of both single probe and multiple probe (such as a double probe) types.
• the open circle nucleic acid probe which has interacted with the target sequence at the duplex regions requires circularisation.
• circularisation is meant the formation of a closed circle. Circularisation may be performed by any one of a number of methods including, but not limited to, gap-filling or spacer oligonucleotide ligation.
• gap-filling is a reference to the circularisation of an open circle nucleic acid probe via the synthesis of a nucleotide sequence to link the terminal ends of the open circle nucleic acid probe.
• the open circle nucleic acid probe is reacted with the required dNTP's, ligase and DNA polymerase.
• spacer oligonucleotide ligation is meant the insertion of one or more previously synthesised nucleotide sequences (referred to as “spacer oligonucleotides”) into the gap between the 5' and 3' ends of the open circle nucleic acid probe. The ends of the spacer are then ligated with the ends of the open circle nucleic acid probe using, for example, the ligase enzyme. Where more than one spacer oligonucleotide is utilised they may be, for example, ligated in tandem to fill the gap between 5' and 3' ends of the open circle nucleic acid probe.
• the reaction mixture will usually comprise, in various ratios, the circularised nucleic acid probe (also referred to as a "closed circle nucleic acid probe"), open circle nucleic acid probes and linear nucleic acid probes.
• the linear nucleic acid probes are those probes which did not interact with or did not ligate to a target nucleic acid sequence.
• any remaining open circle nucleic acid probes will include both those probes which are unaltered by the circularisation step and those probes which were incompletely circularised, for example, where the spacer ligated to only one of the nucleic acid probe ends (ie. either the 5' or the 3' end) or where the gap-fill synthesis was only partially completed.
• the resultant amplification products will include:
• nucleic acid sequence (i) a nucleic acid sequence synthesised from the first primer.
• This nucleotide sequence will comprise tandem repeats of a sequence complementary to that of the closed circle nucleic acid probe;
• nucleotide sequence (ii) a nucleotide sequence synthesised from the second primer.
• This nucleotide sequence will comprise tandem repeats of a nucleic acid sequence complementary to the nucleic acid sequence generated by the first primer.
• amplification products may exist as single stranded nucleic acid sequences or as nucleic acid sequences either completely or partially hybridised to a complementary nucleic acid sequence.
• partial hybridisation is meant that part of the nucleic acid sequence is hybridised to a complementary sequence and part of the nucleic acid sequence is in single stranded form. This will occur, for example, due to the effects of strand displacement such as where primers have interacted with two or more of the tandem repeats of a nucleic acid sequence and the amplification product synthesised from a downstream primer encounters the adjacent upstream primer. In this case, the amplification primer generated from the downstream product will displace the upstream primer as it continues its complementary synthesis extension.
• the inventors have characterized a previously unidentified background amplification product, termed the AmpX reaction, which is also produced.
• This amplification product is usually a nucleotide sequence comprising one or more tandem repeats complementary to the probe sequence but stretching from the first primer region to the second primer region and including the first and second primer regions. These tandem repeats may, however, also comprise extra nucleotide sequence flanking the downstream primer site and/or deletions in the nucleotide sequence.
• This reaction occurs in the presence of open circle or linear nucleic acid probes. The precise mechanism by which this AmpX reaction occurs is unknown, however it is thought to involve some form of illegitimate priming and strand invasion events of the open circle or linear nucleic acid probes.
• the inventors have developed a method of amplifying a circular nucleic acid molecule which method incorporates an enrichment step which is performed following interaction of the nucleic acid probe to the target nucleic acid sequence but prior to the amplification of the probe.
• the enrichment step may be performed either before or after the formation of closed circle probes.
• references to “enriching” should be understood as a reference to increasing the ratio of closed circle nucleic acid probes relative to the linear nucleic acid molecules. This can be achieved, for example, by degrading, removing, inactivating or otherwise reducing the linear nucleic acid molecules (such as linear nucleic acid probes and/or linear target sequences) or by specifically isolating the closed circle nucleic acid probes from the reaction mixture.
• Enriching for closed circle nucleic acid probes can be achieved by any one of a number of methods including, but not limited to, electrophonetic separation, chromatographic separation (for example by size exclusion or affinity chromatography) or degrading the linear nucleic acid molecules utilising, for example an enzyme such as an exonuclease (referred to herein as "enzymatic enrichment").
• exonucleases function by cleaving the terminal nucleotides from a linear nucleic acid molecule. Closed circle nucleic acid probes are not degraded and thereby undergo enrichment.
• linear and/or open circle molecules may be digested utilising the enzyme exonuclease III which functions by degrading free DNA termini but does not degrade closed circle molecules.
• This step enriches for closed circle molecules by selectively removing linear and/or open circle molecules and is preferably performed after the circularization step but prior to the amplification step. Enzymatic enrichment is particularly useful for achieving enrichment of closed circle nucleic acid probes by reducing the population of linear and/or open circle nucleotide sequences.
• the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enzymatically enriching generation of a circular nucleic acid probe and enzymatic enrichment for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
• the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic emichment; and subjecting said enriched circular nucleic acid probe to amplification.
• said enzymatic emichment is performed utilising an exonuclease.
• closed circle nucleic acid probes can be enriched for utilising non-enzymatic methods.
• non-enzymatic methods suitable for use in the method of the present invention include, but are not limited to, electrophonetic separation, chromatographic separation (for example by size exclusion or affinity chromatography) or the introduction of a capture ligand into the closed circle probes via which the closed circle probes can thereby be isolated.
• electrophonetic or chromatographic separation may be designed, for example, reduce the proportion of linear nucleic acid molecules while the use of a capture ligand is particularly useful for facilitating the isolation of closed circle nucleic acid probes.
• the capture ligand may be introduced, during circularisation, into the region intervening the terminal ends of the open circle probe.
• the present invention is not limited to the introduction of a capture ligand by this particular method.
• the capture ligand may be introduced into other regions of the nucleic acid probe such that it facilitates the isolation of closed circle nucleic acid probes.
• the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising non-enzymatically enriching for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
• the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic emichment; and subjecting said enriched circular nucleic acid probe to amplification.
• the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• capture ligand is meant a molecule which permits the selective isolation of a nucleic acid probe into which it is incorporated. It may be incorporated by any suitable means.
• the capture ligand may take the form of modified nucleotides which are used to link the 5' and 3 ' terminal nucleotides of the open circle nucleic acid probe (by either gap-filling or spacer oligonucleotide ligation, for example) or it may comprise unmodified nucleic acids (such as a nucleic acid tag), the sequence of which facilitates isolation of probe molecules incorporating the nucleic acid tag.
• the capture ligand may be a nucleotide sequence which comprises the GCN4 recognition sequence (refer Figure 2).
• a nucleotide sequence capture ligand may be introduced which sequence permits the isolation of closed circle nucleic acid probes by differential hybridisation potential.
• this method of enrichment is schematically depicted in Figure 3. In this example, the emichment step is achieved via solid phase capture. However, it should be understood that this method is not limited to solid phase capture.
• the capture ligand may therefore itself both permit selective purification and act to circularise the open circle probe.
• the capture ligand may be an oligonucleotide comprising nucleotide analogues which are ligated into the intervening region.
• the oligonucleotide acts to circularise the open circle probe and by virtue of the modified nucleotides of which it is synthesised, permits selective purification of the probe by virtue of the modification.
• the nucleotide analogues may be introduced into the reaction mixture comprising the open circle probes as dNTP analogues which by gap-fill synthesis circularise the open circle probe.
• the capture ligand may alternatively take the form of a nucleic acid molecule or a nucleotide to which a capture molecule is linked, bound or otherwise associated which nucleic acid molecule or nucleotide will link the 5 ' and 3 ' terminal nucleotides of the open circle nucleic acid probe.
• the present invention should be understood to extend to the use of any suitable molecule to comprise the capture ligand via its association with one or more linking nucleotides.
• magnetic beads which are coupled to a gap-fill oligonucleotide are envisaged as are molecules such as a hapten which can be bound by an antibody.
• the capture ligand is one which is resistant to the denaturing conditions which are applied to the reaction mixture to achieve breaking of the hydrogen bonds of the duplexes.
• This step is usually performed to free open circle nucleic acid probes which may co-purify with the target molecules during the emichment step.
• the capture ligand comprises a biotinylated oligonucleotide. Following ligation of this oligonucleotide into the open circle nucleic acid probe a closed circle nucleic acid probe is formed. The closed circle probe can be isolated by binding the biotin molecule which is coupled to the ligated oligonucleotide to streptavidin.
• Enrichment of the closed circle probes incorporating a capture ligand may be achieved by any suitable method such as, but not limited to, the cross linking and precipitation of the closed circular nucleic acid probes comprising the capture ligand or coupling of the closed circular probes to a solid phase via the capture ligand.
• one embodiment of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a biotinylated capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the circular probe may be subjected to amplification according to methods well known to those skilled in the art. Without limiting this aspect of the present invention in any way, amplification may be performed by initiating nucleotide extension from a primer complementary to a portion of the circular probe. Through the use of strand-displacing DNA polymerases this extension reaction produces large tandemly arranged multimeric single-stranded DNA products, complementary to the circular target. This occurs due to continual displacement of any nucleic acid downstream of the rapidly extending 3' end of the new strand.
• This reaction in itself allows linear amplification of DNA from the circular probes but the size of the molecules produced and the level of amplification obtained is limited by the processivity of the strand-displacing DNA polymerase.
• a second oligonucleotide primer is used to achieve exponential amplification kinetics under isothermal conditions.
• the multimeric polymers that are produced from the initial priming events on the circular probes comprise multiple primer binding sites for this second primer, thereby facilitating the simultaneous initiation of multiple DNA strand synthesis.
• the resultant product is a network of highly branched strands elongating and displacing down the length of the multimeric polymers.
• the original primer complementary to the circular probe can also prime these displaced strands. This molecular cascade then continues until there are no more primable sites or until one of the substrates for the reaction is depleted. It is also thought that the completely displaced strands themselves may act as primers by interacting with other displaced strands. This results in significantly greater amplification than is obtainable by traditional nucleic acid amplification techniques.
• the method of the present invention does not necessarily selectively isolate only closed circle nucleic acid probes from the reaction mixture. Rather, it is a method for enriching for closed circular probes. For example, where a circularised probe is in a padlock conformation around a target sequence, isolation of the probe may also isolate the target sequence due to the padlock conformation.
• the amplification products may optionally be detected using a wide variety of techniques including, but not limited to, staining of the products with intercalating dyes, the incorporation of detection tags directly into the products or they can be coupled with a variety of other detection molecules which are known to those skilled in the art. These could include, but should not be limited to, radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies and ligands.
• the detection of amplified products could include the solid phase - based amplification using rolling circle amplification.
• one of the amplification primers is coupled to a solid support.
• This solid support may be any solid material to which oligonucleotides can be coupled. Such materials are known to those skilled in the art. These materials may be incorporated into multiple formats which include but shall not be limited to magnetic beads, micro titre trays, membranes and dipsticks.
• the second primer used in the amplification reaction contains a molecular tag (e.g. fluorescein).
• a molecular tag such as biotin, DIG or a fluorophore may be incorporated in the form of labelled nucleotides during the synthesis of amplified DNA.
• a molecular tag such as biotin, DIG or a fluorophore
• some of the products will be coupled directly to the surface of the solid support.
• these products will be labelled due to priming from the second primer containing the molecular tag. Unused primers and other by-products of the reaction can therefore be directly washed from the solid support without disrupting the attached amplified products.
• the attached products can then be identified utilising the molecular tag.
• Primers used in one example of this amplification are schematically depicted in Figure 4.
• the present invention should be understood to extend to the application of rolling circle amplification in the context of DNA microarrays.
• the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe.
• the present invention provides a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment.
• the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment.
• the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; and generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe.
• capture ligand is a biotinylated nucleotide.
• the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and emiching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and emiching for said circular nucleic acid probe by enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
• the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and emiching for said circular nucleic acid probe by non-enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
• the present invention provides in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and emiching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• capture ligand is a biotinylated nucleotide.
• nucleic acid sequences of the present invention may be derived from the human genome but genomes and nucleotide sequences from non-human animals and plants, microbes (for example, bacteria, parasites or yeast), viruses and prion sequences are also encompassed by the present invention.
• Non-human animals contemplated by the present invention include primates, livestock animals (eg. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (eg. mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals (eg. dogs, cats), birds (eg.
• the process of the present invention may be homologous or heterologous with respect to the species from which the nucleic acid molecules are derived.
• a “homologous” process is one where all the nucleic acid molecules utilised in the method of the present invention are derived from the same species.
• a “heterologous” process is one where at least one of the nucleic acid molecules is from a species different to that of other of the nucleic acid molecules.
• any given nucleic acid molecule (such as the nucleic acid probe) will not have been derived from any species but will have been designed to comprise a sequence of nucleotides which are not naturally occurring. Individual regions of the probe may be based on naturally occurring sequences derived from one or more species (for example a promoter region or a target probe region).
• the method of the present invention is useful for improving the specificity of isothermic amplification. This includes, for example, improving the specificity of the generation of tandem compliments of the closed circle nucleic acid probe sequences that are generated by strand displacement synthesis.
• the present invention is also useful in diagnostic applications such as the detection, identification, quantitation and/or typing of specific genetic sequences found in biological or environmental samples such as molecular sequences from human, animal, plant, parasite, bacterial or viral origin.
• the present invention is useful with respect to the diagnosis of genetic or infectious diseases such as bacterial and viral infections.
• one application of the present invention is the probing of biological samples (such as blood, urine, mucus or biopsy specimens) to detect the presence of bacteria or virus wherein the bacterium or virus comprises the target nucleic acid sequence to which the target probe regions of the nucleic acid probe are directed.
• yet another aspect of the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and emiching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• said emiching step is non-enzymatic emichment.
• said enriching step is an enzymatic enrichment step.
• the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and emiching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
• Said target molecule may be present in a biological sample. Accordingly, the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing. It is within the scope of the present invention for the target nucleic acid sequence to be pre-treated prior to testing, for example, inactivation of live virus.
• the method of the present invention is also useful for generating nucleic acid products such as, but not limited to, dendrimeric probes, molecular weight markers, immobilised ligands for affinity chromatography of transcription factors and products for use in the functional analysis of transactivating factors and Southwestern blot analysis.
• nucleic acid products such as, but not limited to, dendrimeric probes, molecular weight markers, immobilised ligands for affinity chromatography of transcription factors and products for use in the functional analysis of transactivating factors and Soiled blot analysis.
• Another aspect of the present invention is directed to a method of amplifying circular nucleic acid probes by designing these probes such that no two primers bind to any one oligonucleotide. Accordingly, no purification of the ligation reaction is necessary.
• a suitably designed nucleic acid probe and primers are schematically illustrated in Figure 19.
• Still yet another aspect of the present invention is directed to a kit for facilitating amplification of a circular nucleic acid probe said kit comprising compartments adapted to contain any one or more of nucleic acid probes, enzymes, capture ligands, means for isolating circular nucleic acid probes and reagents useful for facilitating circularisation, isolation and amplification of said probes. Further compartments may also be included, for example, to receive biological samples.
• said amplification is rolling circle amplification.
• E.coli strain PNG801 is a derivative of the E.coli K12 wildtype strain W1485 (obtained from N. Kleckner, Harvard University). The mini-transposon TnlO (No. 103), encoding a kanamycin resistance gene was introduced into E.coli strain W1485 (10). From the resulting kanamycin resistant poolate one strain was selected and named PNG801. E.coli DH5 which has been described previously (Gibco, BRL) was used as a negative control, representing an E.coli genome not containing the mini-transposon. Genomic DNA was extracted from both E.coli strains (11) and resuspended in TE buffer.
• Oligonucleotides were purchased from Bresatec and Gibco BRL and synthesized using standard phosphoramidite chemistry. Oligonucleotides used as templates in the amplification reactions were gel purified to homogeneity while all others were supplied as desalted preparations.
• Amplification was initiated by the addition of 4 U of Bst DNA polymerase (New England Biolabs) and the reactions were isothermally maintained at 60°C for 2.5-3 hours.
• Amplified products were electrophoresed through 2% v/v agarose gels in TAE or TBE buffer (12) and visualized by ethidium bromide staining.
• nucleic acids Prior to blotting, nucleic acids were denatured in 0.5 M NaOH, 1.5 M NaCl for 30 minutes, followed by neutralization in 1 M Tris-HCl pH 8.0, 1.5 M NaCl for 30 minutes. Nucleic acids were then capillary transferred to Hybond-N + membrane (Amersham) according to the manufacturer's protocol. Oligonucleotide probes were 3' labeled with DIG-ddUTP using terminal transferase (Boehringer Mannheim).
• Membranes were prehybridized in 5-10 ml of RapidHyb buffer (Amersham) at 42°C for 30 minutes. The DIG-labeled oligonucleotides were then added to the hybridization buffer and hybridization at 42 °C overnight. The blots were washed and developed using CDP-Star (Boehringer Mannheim) as per the manufacturer's instructions.
• Products of the amplification reactions were purified through Wizard PCR DNA purification columns (Promega) prior to cloning. The purified products were then ligated directly into the pGEM-T vector (Promega) at 16°C for 3 hours, followed by electroporation into E.coli DH5 (13). Inserts were sequenced using dye termination chemistry and an Applied Biosystems 373 A DNA sequencer.
• a synthetic 90 mer oligonucleotide of random sequence was synthesized and used as a template in initial amplification reactions (Fig. 5 A).
• a variety of smaller primers were also synthesized, based on the sequence of the template molecule. These were designed such that they varied in size, orientation and position with respect to the template molecule (Fig. 5A).
• Various combinations of opposing primers were used in amplification reactions. Typically, 1 pmol of template was mixed with 16 pmol of each primer. Following denaturation and equilibration to 60 °C, the reactions were initiated by the addition of the strand-displacing, exonuclease minus Bst DNA polymerase. The reactions were then incubated at 60 °C for 3 hrs before products were examined by agarose gel electrophoresis (Fig. 5B). A population of products was produced from each of these reactions which had characteristic banding patterns, regardless of the primer combinations tested. The sizes of these products ranged from less than 100 nucleotides, to molecules that were so large that they remained in the wells following electrophoresis.
• Each of the products from a single amplification reaction appeared to differ in size from one another by a standard unit length. This unit length varied between different primer combinations utilized in the reactions. The level of DNA amplification also varied between different primer combinations, but in general, spectrophotometric assays indicated that the reactions were able to synthesize between 10 and 40 ⁇ g of products during a 3 hour reaction.
• amplification products from reactions where primers were situated away from the ends of the template molecule consisted of tandem repeats of a region of the template molecule which spanned from one primer binding site to the next.
• several repeats also included template sequence flanking the primer binding sites.
• the amplification products consisted of tandem repeats of a region of the template molecule as described above but including deletions. Occasionally, sequence deletions vary from one repeat unit to the next. Examples of some of the amplified regions are outlined below.
• reaction products consist of a mixed population of molecules, the majority of which are arranged as tandem repeats of the entire template, and some of which have point mutations in the Sau3AI site.
• Products from the amplification reaction using primers B4I and P5D were digested only with EcoRI and Taql, but not with Sau3AI or Alul (Fig. 6C(i)). This was unexpected as the Alul site is located within the sequence of primer P5D. Sequence analysis indicated that the products from this reaction contained tandem repeats of a region which included the B4I primer binding site and the intervening sequence of the template molecule between P5D and B4I but included only the last 1 1 nucleotides of the primer P5D (Fig. 6C(ii)). The Alul site is within the deleted region of P5D, thus explaining the inability o ⁇ Alul to cleave the reaction products.
• the synthetic template oligonucleotide was diluted using 10 fold serial dilutions to determine the sensitivity limit of the amplification reaction.
• primers B4C and P5C in a 3 hour amplification the detection sensitivity of the reaction was approximately 10 "4 picomoles of template.
• the detection sensitivity of other primer combinations and templates was similar, however, on several occasions the detection sensitivity increased to less than 10 "8 picomoles of template (Fig. 7). This indicates the need for at least lxl 0 8 copies of the template to initiate the reaction. Longer incubations generally did not increase the detection sensitivity of the assay.
• Amplification of 30 ⁇ g of product from lxlO "4 pmol of template represents a potential lxl 0 7 fold amplification.
• Duplicate reactions were set up using various amounts of the genomic DNA from both TnlO positive and negative E.coli strains. Following denaturation and Bst polymerase addition, standard AmpX amplification reactions were carried out using isothermal conditions at 60 °C for 2.5 hours. Following amplification 1 ⁇ L of each reaction was digested with Hindlll (Fig. 8A).
• the population of products amplified in each of the reactions were similar to those observed using the oligonucleotide template. They ranged in size from just over 100 base pairs to extremely large molecules. Each of the products from a single amplification appeared to differ in size from one another by a standard unit length. However, in this example the unit length varied quite dramatically from reaction to reaction even though the primer combination remained unchanged. This was thought to possibly represent variations in the initiation events of the reaction that led to this amplification occurring. Digestion of the reaction products with Hindlll indicated that the majority of the products from each of the reactions contain a uniform repeat size. Furthermore, the labelled internal probe, In903, was able to hybridise with all reaction products verifying the presence of that region of the intervening sequence in the reaction products.
• Sequence analysis of products from three separate amplification reactions revealed different sets of tandem repeat units within each of the populations, as expected. Each contained both primer binding sites and the corresponding intervening sequence but differed in the length of sequence flanking each of the primer binding sites. The length of this additional sequence varied between none to 77 nucleotides. The sequence of the repeat unit did not appear to change between individual repeats in the molecules sequenced. In some experiments with the E.coli PNG801 genomic DNA template, where multiple reactions were performed using a single primer set and a single template concentration, several of the identical reactions failed to amplify. This and the evidence of different amplified regions tends to suggest that specific initiation events need to take place for the reactions to proceed.
• a 50 ⁇ L reaction volume contained 1 pmol of padlock oligonucleotide, 1 pmol of spacer oligonucleotide and 1 pmol of synthetic oligonucleotide target in ligation buffer (20mM Tris-HCl, pH 8.3, 25mM KCl, lOmM MgCl 2 0.5mM NAD, 0.01 % v/v Triton X-100).
• the reaction was initially heated to 94 °C for 3 minutes. Once the tube had reached 94 °C, l ⁇ L of Ampligase (5U/ ⁇ L; Epicentre Technologies) was added. The reaction was then cooled to 60 °C and incubated for 1 hour to allow the ligation to take place.
• a 50 ⁇ L reaction volume contained 1 pmol of padlock oligonucleotide, 1 pmol of synthetic oligonucleotide target, 15 pmol of biotin- 14-dATP and 15 pmol of each dGTP, dTTP and dCTP in ligation/gap-fill buffer (50mM N-(2-hydroxyethyl) piperazine-N-(3- propanesulfonic acid) [EPPS], 180 mM K + (KOH added to adjust pH to 7.8 and KCl added for final K + concentration), 10 mM MgCl 2 , 10 mM NH 4 C1, 100 ⁇ M NAD + , 100 ⁇ g bovine serum albumin (BSA).
• ligation/gap-fill buffer 50mM N-(2-hydroxyethyl) piperazine-N-(3- propanesulfonic acid) [EPPS]
• 180 mM K + KOH added to adjust pH to 7.8 and KCl added for final
• the reaction was initially heated to 94 °C for 3 minutes. Once the tube had reached 94°C, l ⁇ L of enzyme mixture (Ampligase 5U/ ⁇ L; Epicentre Technologies & Taq polymerase lU/ ⁇ L; Perkin Elmer) was added. The reaction was then cooled to 60 °C and incubated for 1 hour to allow the ligation and gap-filling reactions to take place.
• enzyme mixture Ampligase 5U/ ⁇ L; Epicentre Technologies & Taq polymerase lU/ ⁇ L; Perkin Elmer
• the amount of molecular tag should not exceed the maximum binding capacity of the affinity substrate medium used in the purification procedure.
• a 60 ⁇ L reaction contained 10 pmoles of each amplification primer, 167 ⁇ M dNTPs, and l ⁇ L of ligation reaction (unpurified or purified) in 20mM Tris-HCl pH 8.8 (25 °C), lOmM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1 % v/v Triton X-100.
• the reactions were heated at 94 °C for 3 minutes before being cooled to the desired amplification temperature (50-70°C) for 5 minutes.
• 0.5 ⁇ L of Bst DNA pol. (8U/ ⁇ L; NEB) was added to each of the reactions and the tubes were incubated at the desired amplification temperature for 1 Vi - 2 hours.
• Time can be increased of decreased depending on the amount of circle present and the efficiency of the particular amplification reaction.
• RCA rolling circle amplification
• Fig. 10 Two standard types of banding are typically evident (Fig. 10).
• template is present and in sufficient concentration a typical RCA banding pattern is seen as a ladder of DNA molecules that differ in size from one another by a standard unit length (Fig. 10 unpurified and purified +ve lanes). Accordingly, this unit length corresponds to the size of the circular molecule utilized as the target in the RCA reactions.
• Fig. 10 unpurified and purified +ve lanes
• the products are arranged as a ladder of DNA molecules that differ in size from one another by a standard unit length but this unit length does not correspond to the size of the expected circular molecule.
• the size of the repeat is typically the same size as the intervening region between the two primers (Fig. 10 unpurified -ve lane).
• This background reaction has been termed "Amp X". It has also been demonstrated that this background reaction is due to remaining uncircularized padlock probe that remains in the reaction following ligation.
• Utilizing tagged spacer molecules in closed circular probe generating reactions with padlock oligonucleotides it is possible to incorporate the tagged molecules into the closed circular probes. These tagged molecules can then be purified using an affinity between the molecular tag and the substrate therefor. Through subsequent washing steps under both non-denaturing and denaturing conditions templates for the RCA reactions which are free from this Amp X background, can be generated (Fig 10 purified - ve lane).
• Fig. 11 Utilizing the closed circular probe generation (tagged spacer) and purification protocols, the versatility of the new RCA procedure was tested using sequences shown in Fig. 11.
• the negative control reaction (Fig. 12 lane 1) contained Padlock FV2 and LigW but did not contain any template nor was any ligase added to the reaction in subsequent steps.
• Homozygous wildtype human gDNA (770ng) was used as a template in ligation reaction with Padlock FV2 and LigW (Fig. 12 lane 2).
• Two other positive control ligation reactions was also setup; a wildtype control reaction (Fig.
• the unpurified and purified ligation reactions were then used as templates for RCA, again using primers FV3 and FV4 in amplifications at 60°C for 1 hour 40 minutes. Only l ⁇ L of each ligation was added to RCA reaction which equates to the equivalent of 15.4 ng of human gDNA or 20 fmol of oligonucleotides being used as templates for the respective ligation reactions.
• An RCA reaction was also done without the addition of template to determine whether any background present was due to the amplification primers FV3 and FV4 (Fig. 12 lane C). A lO ⁇ L aliquot of each reaction was electrophoresed through 2% agarose in TBE buffer, alongside ⁇ X174 Haelll digested DNA marker (Fig.
• Example 7 it was possible to detect a sequence of interest from as little as 15.4 ng of human gDNA using RCA reactions.
• the sensitivity of RCA has been further tested on purified circular molecules. Specifically, untagged circular molecules have been purified by excising the correct band from ligation reactions, using synthetic target molecules, run out on denaturing polyacrylamide gels. These circular molecules were extracted, purified and quantitated using UV spectrophotometric analysis. Dilutions of this purified material were then used as templates in RCA reactions (Fig. 13).
• the reaction was able to detect fewer than 10 circular molecules present in the RCA reactions. Furthermore, if these reactions are slightly modified by the addition of 15 ⁇ g of bovine serum albumin and l ⁇ L of Sybr Green (1 :1000 dilution; Molecular Probes) to the reaction buffer, the reactions can be followed using real time fluorescence measurements to estimate the amount of DNA generated (Fig. 14).
• a threshold fluorescence level can be chosen and when the time taken for each sample to reach this threshold is plotted against the log of the amount of circular molecules present a linear relationship is observed (Fig. 15).
• RCA reactions are quantitative and allow prediction of the number of circles added to an amplification reaction.
• RCA is able to differentiate single nucleotide polymorphisms (SNPs) when they are incorporated into the closed circular probes.
• Wildtype and mutant circles were generated in ligation reactions using Padlock FV2 with either LigW and wildtype target oligonucleotide (lpmol) for generating wildtype circles (W) or LigM and mutant target oligonucleotide (lpmol) for generating mutant circles (M) (see Figures 11 and 17).
• a negative control reaction to which no template was added nor ligase added during subsequent steps was also included (C). Ligations were carried out according to the standard protocol at 60 °C for 1 hour and circles were then purified using the standard purification protocol.
• the amplification primers require careful design. Generally one primer is designed to a region of the closed circular probe that represents the "backbone" of the padlock probe.
• the amplification specific primers must be designed with their 3' end adjacent to the mutation specific base. It is also thought that mutations in the oligonucleotide downstream to the last base on the 3' end of the primer will also help in differentiation during amplification.
• the primers chosen for SNP detection with respect to the factor V gene are illustrated in Figure 17.
• This example involves discriminating between synthetic DNA targets derived from Chlamydia trachomatis and Chlamydia pneumoniae:
• the targets were 40 base single strand synthetic fragments of the C. trachomatis and C. pneumoniae. groEL gene which encodes heat shock protein 60 (HSP60).
• a single padlock probe was used that annealed to conserved sequences, leaving a gap that included species specific sequence.
• primers were designed as follows.
• Primer FVCOMT is antisense with respect to the padlock probe and is designed to anneal to the padlock backbone ie. , the part of the padlock probe that does not anneal to the target. It also contains an oligosaccharide-T domain at its 5 ' end to act as a spacer between it and the solid support.
• the allele specific primers are in the same sense as the padlock probe and spacer sequence and are designed such that the 4 bases at the 3 ' end are the same as the 4 bases at the 5' end of the spacer sequences.
• the ligation consisted of 1 X T4 DNA ligase buffer (Life Technologies), 6 pmoles padlock HSP2, 52 pmoles Pne spacer, 52 pmoles Tra spacer, 3.2 U T4 DNA ligase (Life Technologies) and 50 pmoles synthetic target in a 40 ⁇ L reaction. Ligations were 10 performed at room temperature for 20 minutes after a 3 minute denaturation at 94°C.
• thermopolymerase buffer New England BioLabs
• RCA Solution was removed from wells by washing 3 X with TBST at room temperature (lOOmM TRIS-HCI pH 7.5, 150 mM NaCl, and 0.1 % v/v Tween 20). Wells were blocked with 50 ⁇ L lOmg/mL BSA for 30 minutes and washed 3 X with TBST. Anti-
• the coating mix contained 100 nM FVComT, lOmM EDC (l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide) and lOmM 1-methyl-imidiazole. To each well lOO ⁇ L was added and the wells were sealed and incubated at 50°C overnight. Wells were washed 3 X TBST, then soaked for 5 minutes and washed with 3 X TBST. To remove residual salt, wells were washed once with deionised water and stored at 4°C.
• Oligonucleotide sequences C. pneumoniae derived synthetic target: TCCTTAACTTTCTATAATCTGC AAACTAGTATTTTATTTTAGGACGGCC ATG ⁇ 400 >27
• SPACER TRA 5'6bT GCA AAC3' Padlock probe (LOCKHSP2): 5'6GC AGG TAA AGA AGG CGC CGC GGT GAG CTA TAT GGG GAC TAT GAA TTT GCT CCA TTA AAG CAA ATT GC3' ⁇ 400 > 30
• C. pneumoniae specific primer PNE4: 5'7CC ATT AAA GCA AAT TGC AAG T3 ' ⁇ 400 > 30
• TRA4 trachomatis specific primer
• Padlock Circularisation Utilizing the closed circular probe generation (tagged spacer) and purification protocols the ability of rolling circle amplification to initiate from primers immobilized on solid supports was tested. Both positive and negative control ligations were setup. The negative (-ve) and positive (+ve) control reactions both contained 1 pmol of each Padlock FV2, Lig W and wildtype target oligonucleotides. Both reactions were heated to 94 °C and then 1 ⁇ L of Ampligase was added to the positive control reaction only. The reactions were cooled to 60 °C and incubated for 1 hour to allow ligation to take place. Following ligation both reactions were purified by the outlined protocol. Padlocks and oligonucleotide sequences are described in Figure 4.
• FVComT primer (5 pmol) was covalently linked to ⁇ ucleolink plates ( ⁇ unc) according to the manufacturers instructions. The plates were blocked in 1 % w/v BSA (100 mg/mL) in TBST buffer (100 mM Tris HCl pH 7.5; 150 mM ⁇ aCl; 0.05% v/v Tween-20) by incubated for 30 minutes at room temperature. The blocking solution was subsequently removed from the plates and amplification reactions assembled in the wells. Standard 60 ⁇ L reactions were setup each containing 10 pmol of fluorescently labelled FVW1 primer. Various amounts of additional FVComT primer were also added to the well for the positive control reactions but in solution rather than attached to the wells.
• a single reaction containing 10 pmol of FVW1 and 10 pmol of FVComT in addition to primer bound to the wells was used to test the negative control reaction.
• l ⁇ L of the respective templates was added to each reaction and the wells were heated at 94 °C for 3 minutes and then cooled to 60°C.
• Bst polymerase (4 units) was added and the wells were incubated for 100 minutes at 60°C.
• C.pneumoniae specific primer -ve 1 No target, C.pneumoniae specific primer -ve 2: C.pneumoniae target, C. pneumoniae specific primer, no ligase Pne+ : C. pneumoniae target, C.pneumoniae specific primer Pne-: C. pneumoniae target, C. trachomatis specific primer Tra+: C. trachomatis target, C. trachomatis specific primer Tra-: C. trachomatis target, C. pneumoniae specific primer Blank: colour development reagents only.

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