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WO1998015652A1 - Sequençage d'acide nucleique par ligature d'adapteurs - Google Patents

Sequençage d'acide nucleique par ligature d'adapteurs Download PDF

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Publication number
WO1998015652A1
WO1998015652A1 PCT/GB1997/002734 GB9702734W WO9815652A1 WO 1998015652 A1 WO1998015652 A1 WO 1998015652A1 GB 9702734 W GB9702734 W GB 9702734W WO 9815652 A1 WO9815652 A1 WO 9815652A1
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Prior art keywords
sequence
adaptor
sequencing
fragments
nucleic acid
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PCT/GB1997/002734
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English (en)
Inventor
Gunter Schmidt
Andrew Hugin Thompson
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Brax Genomics Limited
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Priority to AU45663/97A priority Critical patent/AU4566397A/en
Publication of WO1998015652A1 publication Critical patent/WO1998015652A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry

Definitions

  • the invention relates to a method for sequencing nucleic acid, especially DNA.
  • modified nucleotides can be used to terminate polymerase extension of nucleic acid being copied from a template strand.
  • To determine the sequence of a template strand four dideoxynucleotides are needed corresponding to the four normal bases.
  • Template strands are added to a polymerisation medium containing all four normal nucleotides and one of the four dideoxynucleotides, which is labeled, usually with a fluorescent dye.
  • the dideoxynucleotide in each medium is at a concentration such that it has a small but defined probability of being incorporated into an extending copy of the template rather than its corresponding normal nucleotide. This terminates chain extension for this fragment. If all the fragments in a particular medium are separated on a sequencing gel, which resolves nucleic acids to a difference in length of one nucleotide, fragments corresponding to termination at every occurrence of the base to which the dideoxynucleotide corresponds should be observed. If a gel for each base is run then there should be observed a band for each nucleotide in the template and the nucleotide sequence should be determined.
  • the cleavage reagents are, however, not totally specific, so this is a fairly 'noisy' system. It suffers from the same problems as the chain termination method too.
  • the method of Harding and Keller acts on immobilised DNA templates.
  • the templates are single stranded and constructed from analogues of normal bases bearing unique fluorescent labels .
  • the immobilised templates are cleaved with a 5' to 3 ' exonuclease to release bases into a flowing medium that is run through a fluorimeter that detects which base is present at the highest concentration in the medium.
  • the exonucleases cleave off bases simultaneously from each copy of a nucleic acid simultaneously, the signal at any time should correspond essentially to the last base cleaved.
  • the sequence of bases flowing past the fluorimeter thus corresponds to the sequence of the fluorescent template.
  • This sort of approach has a potential to be extremely rapid, limited only by the processivity of the exonuclease used, which could be of the order of 100 to a 1000 bases a second.
  • This method requires base analogs that are distinguishable by some means from each other, preferably by fluorescence. These analaogs must be incorporated into a template as normal bases and be cleaved by an exonuclease as normal bases. Alternatively polymerases and exonucleases must be engineered to recognise the analogs. Either way is a major technical obstacle. Furthermore, the requirement for simultaneity of cleavage of corresponding bases from a population of immobilised template allows only a small margin of error which will severely limit the length of sequence that can be determined by this approach. These technical obstructions remain to be overcome.
  • Arrays of single-stranded oligonucleotides can be constructed representing for example, every possible combination of the 4 bases in an 8 bp oligonucleotide. Each point on an array would correspond to one such oligonucleotide.
  • a single-stranded template with a fluorescent label can be hybridised to to such an array. Every overlapping linear sequence of 8 bp that is contained in the template will be represented on the array and the template should hybridise to every point corresponding to each 8 bp sequence that defines it . The fluorescence from every point on the array can then be determined and the sequence of the target reconstructed.
  • the present invention provides a method for sequencing nucleic acid, which comprises:
  • the label may be any label suitable for the purpose, such as a fluorescent label or a mass label.
  • Each label may comprise a mass label associated with a corresponding known base sequence for identifying the corresponding _base sequence in mass spectrometry.
  • each adaptor oligonucleotide is labelled with an associated mass label which is uniquely resolvable in mass spectrometry from the other labelled adaptor oligonucleotides.
  • each adaptor oligonucleotide is composed of nucleotide analogues which are resistant to fragmentation in the mass spectrometer .
  • each mass label is cleavably attached to its corresponding adaptor oligonucleotide and uniquely resolvable in mass spectrometry.
  • the mass label may be attached to the adaptor oligonucleotide by a cleavable linker and may be cleaved under any appropriate cleavage conditions such as photocleavage conditions or chemical cleavage conditions.
  • the mass spectrometry may be effected using a mass spectrometer with orthogonal time of flight or array detector geometry.
  • the fragments are contacted in step (i) with the array of adaptor oligonucleotides in a cycle wherein the cycle comprises sequentially contacting each adaptor oligonucleotide of the array with the fragments.
  • the target nucleic acid population is subjected to sorting into sub-populations according to their sticky end sequences and each of the sub-populations is subjected to steps (b) and (c) .
  • each fragment may be produced by differential application.
  • the predetermined length of this base sequence of the sticky ends is from 3 to 5 , more preferably 4.
  • the sequencing enzyme preferably comprises a type IIs restriction endonuclease .
  • the target nucleic acid population may comprise heterogeneous nucleic acid fragments. _ The other end of each fragment may be protected by ligation with an immobilisation adaptor oligonucleotide.
  • FIGURE 1 shows a cloning vector for template sequences
  • FIGURE 2 shows PCR amplification of template DNA
  • FIGURE 3 shows immobilisation of amplified template DNA
  • FIGURE 4 shows a method of differential amplification of template
  • FIGURE 5 shows a method of producing protected DNA fragments with termini for sequencing
  • FIGURE 6 shows the action of Fokl
  • FIGURE 7 shows cutting behaviour of typical adapters according ot the invention
  • FIGURE 8 shows an adapter cycle according to one embodiment of the invention.
  • FIGURE 9 shows graphs of the effects of PEG and Ficol on ligation at ATTA and GCCG.
  • This invention provides a method capable of simultaneously sequencing a heterogeneous population of nucleic acid fragments.
  • the technology is compatible with numerous methods of template preparation.
  • the invention however provides a novel preferred strategy for sequencing large DNA molecules that limits the need for biological sub-cloning hosts and vectors.
  • the sequencing process may be summarised:
  • the sequencing method described here allows one to produce nucleic acid fragment populations in a_ reproducible manner that can then be sorted into subsets and finally sequenced by an oligonucleotide adapter based technique.
  • the sequencing method described requires double stranded templates .
  • the sequencing technique is most effective with immobilisation of the nucleic acid templates at one terminus, the other terminus must be accessible to adapters.
  • the sequencing steps use adapter molecules to generate and probe the sequence of terminal single-stranded overlaps of immobilised nucleic acid fragments.
  • Single-stranded overlaps are generated in a cyclical process preferably through the use of type IIs restriction endonucleases .
  • Recognition sites for these enzymes are provided by adapters at the terminus of a template sequence. The position of the recognition site is arranged so that digestion in the presence of the type IIs endonuclease exposes an ambiguous sticky end in the unknown sequence of the template.
  • the resultant ambiguous sticky ends generated in template sequences are probed as heterogeneous sets and sequence information is determined by measuring the quantity of label detected from correctly hybridised adapters.
  • the sequence of individual fragments is determined by comparing quantities of label for each adapter in each cycle of the sequencing process with quantities derived in previous and subsequent cycles.
  • the invention provides a method for analysing heterogeneous sub- populations of nucleic acids without spatially resolving them. This is achieved by a signal acquisition and signal processing procedure that allows sequences to be identified on the basis of their relative quantities.
  • This process does not require traditional gel methods to acquire sequence information. Since the entire process takes place in solution and is an iterative process, the steps involved could be performed by a liquid-handling robot or a microfluidics system.
  • Type IIs Restriction Endonucleases have the property that they recognise and bind to a specific sequence within a target DNA molecule, but they cut at a defined distance away from that sequence generating single-stranded sticky-ends of known length but unknown sequence at the cleavage termini of the restriction products .
  • the enzyme fokl generates an ambiguous sticky-end of 4 bp, 9 bp downstream of its recognition sequence.
  • This ambiguous sticky-end could thus be one of 256 possible 4-mers. (see figure 6) .
  • a preferred method for use with this invention requires fragmentation of the target nucleic acid followed by molecular sorting into sub-populations that are small enough to allow simultaneous sequencing.
  • This preferred method requires the use of adapters.
  • Two adapter based sorting methods are described here. One requires the use of a type IIs restriction endonuclease or a similar system for generating ambiguous sticky-ends in double stranded DNA while the second uses a primer and DNA amplification based approach to sorting.
  • Nucleic acids may be fragmented in numerous ways which may be either directed or random. For the purposes of sequencing large nucleic acids, an approach which generates numerous fragments that overlap randomly is favoured in conventional sequencing strategies for large nucleic acid templates, due to its redundancy and relative simplicity. Obviously this sort of approach requires multiple copies of the target nucleic acid to ensure that all sequences are represented in the population of fragments with unambiguous overlaps with other fragments.
  • Random fragmentation will work excellently with certain embodiments of this invention but to try and reduce redundant sequencing more controlled fragmentation of a target nucleic acid could be used.
  • a set of relatively high stringency restriction endonucleases can be used to generate sets of overlapping fragments . In this way one would hope to generate overlapping contigs in a more economical manner than random fragmentatio .
  • Random fragmentation can be achieved with mild digestion of the target nucleic acid by DNAsel or sonication. Generally, blunt - ended fragments are generated by this approach.
  • the DNA is first sub-cloned into a library.
  • the process of producing a library of this sort can be done in-house or by commercially available services, such as that provided by Clonetech.
  • the DNA is fragmented (e.g. by restriction enzyme digestion or sonication) to sizes in range of a few hundred bases and then sub-cloned into a cloning vector of choice. Because each fragment in the library is flanked by the same vector sequence a standard set of flanking PCR primers can be used to PCR amplify each fragment. Using the same PCR primers for each fragment also helps to normalise the efficiency of each PCR reaction as primer sequence is one of the most important factors affecting amplification efficiency, (see figure 1)
  • the library is then transfixed into an appropriate bacterial strain and the bacteria plated out onto selective agar plates.
  • Individual colonies are then picked by a colony picking robot (which are commercially available) .
  • Each picked colony is then spiked into a unique PCR reaction, set up on a microtitre plate for example, and each fragment is PCR amplified using the standard primer set which flank the insert .
  • One of the primers used in this reaction must be biotinylated which will allow the subsequent capture of the amplified fragment, (see figure 2)
  • a known amount of each of the amplified fragments can be captured on a streptavidin coated surface by its biotinylated primer.
  • a specific amount of PCR product can be captured. (This does, however, rely on all the primers being incorporated into the amplification products. This should only require a simple primer titration optimisation experiment as PCR reactions using clones are highly efficient.)
  • Different protocols can be used for this purpose, for example streptavidin coated magnetic beads or streptavidin coated wells of a microtitre plate.
  • beads which will bind 1 pmol of biotin per ul of beads
  • adding 5 ul of beads and the appropriate buffer to the PCR reaction will capture 5 pmol of the amplified fragment.
  • the use of beads also allows the capture of different quantities of individual amplified fragments. By adding differing amounts of beads to separate amplification reactions prior to pooling them, one can, for example, create a heterogeneous population with lpmol of fragment 1, 4 pmol of fragment 2, 10 pmol of fragment 3 and so on.
  • streptavidin coated wells of a microtitre plate could also be used by transferring each amplification reaction to a unique well of the microtitre plate.
  • Commercially available streptavidin coated plates usually have a maximum binding capacity of between 5 to 20 pmol of biotin. Therefore, the amount of amplified fragment captured in each well is determined by the binding capacity of that plate.
  • quantification of template must be stringent. This can be achieved by labelling one of the primers. After the amplification reaction has been performed, the biotinylated fragments can be captured on an avidinated substrate and washed. The number of copies of template pres.ent can be determined by measuring the retained fluorescence. Appropriate dilution of the amplified template can be performed if desired before sequencing. This gives an additional level of control over and above the capture steps .
  • the vector sequences at either terminus of the DNA can be designed to bear distinct primer sequences. This would ensure that one terminus can be readily identified as a sequencing terminus and one terminus could be designated as the immobilisation terminus. A unique termination sequence at the immobilisation terminus would identify when the clone had been sequenced.
  • Type IIs restriction endonuclease sites in the immobilisation terminus sequence can additionally permit molecular sorting.
  • the sequencing terminus can be engineered to carry a recognition sequence for the type IIs restriction endonuclease to be used as the sequencing endonuclease.
  • These vector sequences can additionally provide primer sequences to permit amplification of template and amplification based sorting.
  • the precise method of ligation will depend on how the fragments are generated. If fragments of the target nucleic acid are generated by using a type IIs restriction endonuclease, adapters with sticky-ends complementary to subsets of the possible sticky- ends that would be generated by the fragmentation endonuclease, can be ligated to the resultant fragments. These adapters could carry designed primer sites that would allow much greater control of the amplification step. The combinations of subsets of sticky- ends of the primer adapters will determine which subsets of fragments are amplified and how large these subsets of fragments are . This will allow much greater control over the PCR amplification steps. See Figures 4 and 5.
  • the parallel sequencing process described here lends itself to this sort of cloning strategy because of its ability to simultaneously sequence heterogeneous populations without spatial resolution of nucleic acids which conventional sequencing strategies cannot achieve. This means if a set of primers generated more than one fragment, the ability to sequence multiple templates simultaneously would allow one to determine the sequence without having to separate the amplified fragments.
  • nucleic acid can be fragmented initially with the sequencing enzyme. This will generate 3 classes of fragments, one class with the sequencing enzyme recognition site at one terminus only, one class with the sequencing enzyme recognition site at both termini and a third class with the site at neither termini.
  • a complete adapter set i.e. corresponding to all sticky-ends, can be added to the restriction fragment population.
  • the adapter would bear the recognition site for the sequencing enzyme. Addition of the sequencing enzyme to a population of fragments with these adapters can have two results. If a given terminus has a recognition site already then the sequencing enzyme can cleave either at the adapter site or at the more internal site. There is a 50 % chance of either cleavage event occurring. At other sites where there is no internal site, clearly, terminal bases must be lost by this process. Since with each round of this process only half of the internal sites will be removed, the process must be repeated at least 7 times to ensure removal of the sequencing enzyme recognition sequence from at least 99 % of the fragments in the population. Thus fragment size may be significantly reduced if a sequencing enzyme is used that cleaves at a significant distance from its recognition site.
  • the pair of adapters shown can be ligated to a fragment generated by Sau3AI .
  • the first adapter bears a recognition sequence for fokl while the second adapter bears a restriction site for BsuRI .
  • BsuRI is methylation sensitive and generates blun -ended fragments. If one synthesises template DNA with S-methyl cytosine but uses adapters with ordinary DNA, only the adapter will be cleaved by this will leave fragments amenable to blunt end ligation.
  • Adapter 1 provides immobilisation and the recognition site for the sequencing endonuclease. A simple protocol for generating distinct termini would be as follows:
  • the first step is fragmentation of a large number of copies of a large nucleic acid, preferably with an ordinary type II restriction endonuclease to generate known sticky ends, such as Sau3AI .
  • fragments can then be ligated to adapters. If the fragments are treated with ligase in the presence of the two types of adapters above, this will generate fragments of three types: fragments with both ends carrying adapter 1, fragments with both ends carrying adapter 2 and thirdly fragments carrying adapter 1 at one end and adapter 2 at the other. Statistically the third type of fragment will be in the majority.
  • the fragments carrying adapter 1 can be immobilised on a solid phase matrix derivitised with avidin.
  • the fragments carrying adapter 2 at both ends can be washed away and those fragments carrying two immobilisation adapters will also be immobilised.
  • the immobilised fragments can be removed from the solid phase matrix.
  • Biotin/streptavidin interactions can be disrupted by acid.
  • o Fragments that bore both adapters can be captured by the new terminus generated by cleavage of adapter 2. Capture requires a further adapter which can be immobilised allowing fragments with adapter 1 at both termini to be washed away. Alternatively the 'capture' adapter can introduce a primer sequence. Adapter 1 can additionally provide a known primer sequence to permit the captured fragments to be differentially amplified. A more complex protocol which allows molecular sorting could be achieved using adapter 2 to provide a second type IIs recognition site. (BspMI in the example below)
  • cleavage step after immobilisation is performed with this would generate fragments with ambiguous sticky ends at one terminus which can again be captured by adapters complementary to the sticky ends generated but one can select at this stage which sticky ends to capture.
  • An entire array of all possible adapters can be generated to allow all fragments to be captured and isolated.
  • a hybridisation array on a glass surface would allow spatial sorting.
  • An alternative method would use the adapter sequence to perform differential amplification.
  • the 'capture' adapter used above can provide another terminal type IIs restriction endonuclease site. This will allow another set of ambiguous sticky-ends to be generated allowing further sub-sorting until the nucleic acid fragment population is of the correct size for unambiguous sequence determination.
  • This sorting process above generates, for a 4 bp ambiguous sticky-end, 256 sub-populations. This may be generate nucleic acid populations small enough to begin sequencing or further sub- sorting may be necessary.
  • the actual sequencing method is essentially sequencing by hybridisation and can be understood first by explaining it for the case of a single nucleic acid.
  • a single nucleic acid immobilised at one terminus to a solid phase substrate, and which has an adapter at the other terminus bearing the recognition site for the type IIs restriction endonuclease chosen for sequencing. Digestion in the presence of fokl will generate a 4 bp ambiguous sticky-end.
  • an adapter molecule This would be an oligonucleotide carrying a sticky-end with one, known, sequence of 4 bp of the possible 256.
  • the adapter would additionally carry a label, e.g. a fluorescent tag, and a binding site for the desired type IIs restriction endonuclease to be used to sequence the immobilised nucleic acid. If the adapter is complementary to the ambiguous end of the target nucleic acid, it will hybridise and it will then be possible to ligate the adapter to the target. The immobilised matrix can then be washed to remove any unbound adapter.
  • the terminus of the target nucleic acid will carry also a binding site for the sequencing endonuclease that will allow cleavage of the target nucleic acid exposing further bases for analysis and the above process can be repeated for the next 4 bp of the target . This iterative process can be repeated until the entire target nucleic acid has been sequenced.
  • a more effective method of labelling appropriate for use with this invention is 'mass labelling' .
  • Cleavable mass labels are described in patent GB9700746.2. This describes methods for generation and use of labels that are readily detectable in a mass spectrometer. Mass labelling permits the generation of large numbers of labels. This would permit 256 labels to be generated allowing all 256 probes for a 4 base pair overlap to be tested simultaneously rather than sequentially. This has advantages in a hybridisation based sequencing method as a competitive binding system avoids some of the problems of different binding energies of different 4 base sequences.
  • GB 9700746.2 describes tagging of nucleic acid probes with cleavable mass labels.
  • These labels may be cleaved from the probe at various stages in a probing assay, but a preferred method of cleavage is during the ionisation process .
  • various methods are possible. After the exposed sticky ends of a template are probed with labelled adapters one is left, after washing away non-ligated adapters, with a template terminated with a labelled adapter.
  • labels that are photocleavable, thermo- labile or acid labile, for example, which can be removed at this stage and analysed in a mass spectrometer.
  • one can cleave the adapter from the template with the appropriate type IIs restriction endonuclease whose recognition site is provided by the adapter.
  • the cleaved adapter can be analysed in a mass spectrometer and the mass label can be cleaved during ionisation.
  • Non-cleavable mass labels are also appropriate for analysis of the cleaved adapter. One needs only use sufficient labels to resolve adapters with the same mass in the mass spectrum.
  • a short liquid chromatography step with a denaturing solvent would allow the tagged strand to be separated from the untagged strand.
  • HPLC or capillary electrophoresis separations would be appropriate. Such a separation would probably not be necessary, though.
  • Denaturing the cleaved adaptor might be quite desirable, however. After cleavage, both strands of the adaptor will be extended by 4 bp .
  • the probe strand will be extended by 4 unknown bases .
  • the non- probe strand will be extended by 4 bases complementary to the probe overlap of the probe strand, and so will have a known mass, hence is the preferred strand for mass labelling.
  • Certain 4mers have the same composition, GGCC, GCCG, GCGC, etc and need to be resolved as these will all give the same peaks in a mass spectrum. One need only add sufficient mass to resolve these uniquely.
  • Type IIs restriction endonucleases are also known to sometimes cleave at incorrect positions. Such cleavages should also be identifiable with this approach as an extra base or one base too few will give a shifted mass spectral peak. This should again allow improvement in quantitation.
  • the positioning of the recognition site for the sequencing endonuclease in the adapter will determine whether the next 4 bp exposed are the next 4 bp in the sequence. Or they may overlap partially with the last four base pairs thus giving partially redundant information or they may be further downstream missing out a few bases, thus only sampling the sequence of the immobilised target nucleic acid.
  • sequential bases can be exposed with adapter 1 while bases are sampled at intervals by adapter 2.
  • With adapter 3 redundant information is acquired. Adapter nucleic acid is shown in bold while fokl binding sites are underlined) . Whatever spacing is used, the spatial information relating the 4 bp oligonucleotides is retained.
  • redundant sequence data is desirable from the template nucleic acid in order to relate sequence information from each round of sequencing to the last round. This gives a small amount of redundancy, hence adapter 3 in figure 7 below is a preferred adapter construct.
  • this invention also envisions algorithms for analysing such a data matrix.
  • the algorithm attempts to identify a sequence on the basis of its frequency, i.e. a sequence present at a given frequency will have every sub-sequence present at the same frequency.
  • the algorithm searches through each column of the matrix and attempts to resolve label quantities, that may be sums of sequence frequencies into atomic quantities such that the same set of atomic quantities appear in all columns.
  • the algorithm achieves this by comparing label quantities in a given column with those in the all the other columns. A given atomic quantity that appears in all columns is then assumed to correspond to a unique sequence .
  • Ligation of adaptors is another critical aspect of the invention that must be considered.
  • Chemical methods of ligation are known: o Ferris et al, Nucleosides and Nucleotides 8, 407 - 414, 1989 o Shabarova et al, Nucleic Acids Research 19, 4247 - 4251, 1991
  • enzymatic ligation would be used and preferred ligases would be T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, Pfu ligase and Tth ligase.
  • preferred ligases would be T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, Pfu ligase and Tth ligase.
  • References to the literature are given below: o Lehman, Science 186, 790 - 797, 1974 o Engler et al, 'DNA Ligases', pg 3 - 30 in Boyer, editor, 'The Enzymes, Vol 15B' , Academic Press, New York, 1982
  • Protocols for use of ligases can be found in: o Sambrook et al, cited above o Barany, PCR Methods and Applications, 1: 5 - 16, 1991 o Marsh et al, Strategies 5, 73 - 76, 1992
  • restriction endonucleases Numerous type IIs restriction endonucleases exist and could be used as sequencing enzymes for this process . Table 1 below gives a list of examples but is by no means comprehensive. A literary review of restriction endonucleases can be found in Roberts, R., J. Nucl. Acids Res. 18, 2351 - 2365, 1988. New enzymes are discovered at an increasing rate and more up to date listings are recorded in specialist databases such as REBase which is readily accessible on the internet using software packages such as Netscape or Mosaic and is found at the World Wide Web address : http://www.neb.com/rebase/.
  • REBase lists all restriction enzymes as they are discovered and is updated regularly, moreover it lists recognition sequences, isoschizomers of each enzyme, manufacturers and suppliers and references to them in scientific literature.
  • the protocol would be much the same irrespective of the type IIs restriction endonuclease used but the spacing of recognition sites for a given enzyme within an adaptor would be tailored according to requirements and the enzymes cutting behaviour, (see figure n above)
  • Preferred embodiments of the process will use adaptors labeled with fluorescent labels. Detection of fluorescent signals can be performed using optical equipment that is readily available. Fluorescent labels usually have optimum frequencies for excitation and then fluoresce at specific wavelengths in returning from an excited state to a ground state. Excitation can be performed with lasers at specific frequencies and fluorescence detected using collections lenses, beam splitters and signal distribution optics. These direct fluorescent signals to photomultiplier systems which convert optical signals to electronic signals which can be interpreted using appropriate electronics systems.
  • a favourable approach is to synthesise the sample molecule with appropriate isotopes to give a slightly different mass spectrum, for a molecule with the same chemical behaviour.
  • This approach might be less desirable than external standards for use with large numbers of mass labels due to the added expense of finding or synthesising appropriate internal standards but will give better qunatification than external standards .
  • An alternative to isotope labelling is to identify a molecule that has similar but not identical chemical behaviour as the sample in the mass spectrometer. Finding such analogues is difficult and is a significant task for large families of mass labels.
  • the configuration of the instrument is critical to determining the actual ion count itself, particularly the ionisation method and the separation method used.
  • Certain separation methods act as mass filters like the quadrupole which only permits ions with a particular mass charge ratio to pass through at one time. This means that a considerable proportion of sample never reaches the detector.
  • most mass spectrometers only detect one part of the mass spectrum at a time. Given that a large proportion of the mass spectrum may be empty or irrelevant but is usually scanned anyway, this means a further large proportion of the sample is wasted.
  • Mattauch-Herzog geometry sector instruments permit this but have a number of limitations.
  • Sector instruments are organised into distinct regions, 'sectors', that perform certain functions.
  • the ionisation chamber feeds into a free sector which feeds into an 'electric sector' .
  • the electric sector basically 'focusses' the ion beam which is divergent after leaving the ion source.
  • the electronic sector also ensures the ion stream has the same energy. This step results in the loss of a certain amount of sample.
  • This focussed ion beam then passes through a second free area into a magnetic sector which splits the beam on the basis of its mass charge ratio.
  • the magnetic sector behaves almost like a prism.
  • a photographic plate can be placed in front of the split beam to measure the intensities of the spectrum at all positions.
  • With a family of well characterised mass labels one would probably monitor only sufficient peaks to sample all the mass labels unambiguously.
  • array detectors would allow one to simultaneously and continuously monitor a number of regions of the mass spectrum simultaneously, which might be applicable for use with well characterised mass label families.
  • the limit on the resolution of closely spaced regions of the spectrum might restrict the number of labels one might use, though, if array detectors are chosen.
  • SIM selected ion monitoring'
  • the orthogonal time of flight mass spectrometer This geometry that allows for very fast sampling of an ion stream followed by almost instantaneous detection of all ion species.
  • the ion current leaving the source probably an electrospray source for many biological applications, passes an electrode plate perpendicular to the current .
  • This plate is essentially an electrical gate and is used to generate a repulsive potential which deflects the ion current 'orthogonally' into a time of flight mass analyser that uses a reflectron.
  • the reflectron is essentially a series of circular electrodes that generate an increasingly repulsive electromagnetic fieldthat normalises ion energies and reflects the ion stream into a detector.
  • the reflectron is a simple device that greatly increases the resolution of TOF analysers. Ions leaving the ion source will have different energies, faster ions will penetrate the repulsive field further than ions with a lower energy and so will be delayed slightly with respect to the lower energy ions but since they will arrive slightly before the lower energy ions they will enter the TOF at roughly the same time so all the ions of a given mass charge ratio will arrive at the detector at roughly the same time.
  • the electrical gate is 'closed' to deflect ions into the TOF analyser, the timer is triggered. The flight time of the deflected ions is recorded and this is sufficient to determine their mass/charge ratio.
  • the gate generally only sends a short pulse of ions into the TOF analyser at any one time. Since the arrival of all ions is recorded and since the TOF separation is extremely fast, the entire mass spectrum is measured effectively simultaneously. Furthermore, the gate electrode can sample the ion stream at extremely high frequencies so very little sample is required. For these reasons this geometry is extremely sensitve, to the order of a few femtomoles .
  • PCR product Three different PCR products are used to represent 3 different templates at different frequencies.
  • the PCR product used for this are exons 14, 16 and 19 of the anion exchanger (AE1) as these PCRs have already been optimised in our laboratory. These are referred to as AE14 , AE16 and AE19.
  • AE16 will be at half the concentration of AE14 and AE19 will be at one fifth the concentration of AE14.
  • AE14 sequence ccaaagctgggagagaacagaatgccttggttttctgctgcagatcttccaggaccacccactacagaagac
  • FAM - CTAGAGGACGATCGA GGATG . GATC . TTCCAGGACCACC ... GATCTCCTGCTAGCT . CCTAC . CTAG . AAGGTCCTGGTGG ...
  • FAM - CTAGAGGACGATCGA GGATG . GATC . TGAGACTCCAGGAATAT . GATCTCCTGCTAGCT . CCTAC . CTAG .ACTCTGAGGTCCTTATA...
  • FAM - CTAGAGGACGATCGA GGATG . GATC .ATCTGCCTGGCAG . GATCTCCTGCTAGCT . CCTAC . CTAG . TAGACGGACCGTC .
  • FAM - CTAGAGGACGATCGA GGATG . GATC . TTCCA
  • FAM - CTAGAGGACGATCGA GGATG. GATC. TGAGA
  • the cleaved fragments are then captured, through ligation, to 3 different wells of a microtitreplate each containing a specific adaptor simulating the first cycle of a sequencing reaction, providing the first 4 bases. See below for full sequences
  • TCT N-CGTCG .
  • GTCC GTCC
  • N is a number of bases
  • TCT CAGGACCTTCTAG .
  • Biotin-N-GCAGC AGA. GGAGTCTCAGATC . CATCC .AGCTAGCAGGAGATC
  • N-CGTCG N-CGTCG .
  • TCT CCTCAGAGTCTAG .
  • GTAGG TCGATCGTCCTCTAG -FAM
  • N-CGTCG N-CGTCG .
  • TCT GTCCGTCTACTAG .
  • GTAGG GTAGG .
  • concentration can be measured through fluorescence of the FAM label and the first 4 bases (XXXX) determined. Successful ligation, measured by fluorescence therefore provides concentration information and the first 4 bases of each fragment .
  • the 'Bbv" adaptors were bound to black, streptavidin coated 96 well microtitre plates (Boehringer Mannheim) . This was achieved by incubating lOpmol of the appropriate adaptor in 35ul of lxTE+0. IM NaCl in each well overnight at 4°C. Following the overnight incubation each well was washed 3 times with 50ul of lxTE+O.lM NaCl. The lxTE+O.lM NaCl was removed and 50ul of Ixligase buffer was added to each well and the plate was stored at 4°C untill used.
  • BioFAMFok adaptor was bound to 8 wells by incubating lOpmol of the adaptor in 25ul of lxTE+O.lM NaCl in each well overnight at 4°C. Following the overnight incubation each well was washed 3 times with 50ul of lxTE+O.lM NaCl. A dilution of BioFAMFok (5, 2.5, 1.25, 0.675, 0.3375pmol) diluted in lxTE+0. IM NaCl was added to a series of well and the fluorescence of the plate read in a Biolumin Microtiter plate Reader (Molecular Dynamics)
  • the 3 PCR products used to represent sequence templates at different concentrations were exons 14,16 and 19 from the human erythrocyte anion exchanger gene located on chromosome 17q21-22. Primer sequences use to amplify exons 14,16 and 19
  • biotin into one of the primers in each set will allow their capture to streptavidin coated beads (Dynal UK) .
  • reaction mix was heated at 65oC in a Techne Dryblock for 20 minutes to inactivate the enzyme.
  • DynaBeads M280 will bind 60-120 pmol of biotinylated double stranded DNA.
  • 300ul of DynaBeads M280 at lmg/ml were washed with lOOul TES by holding the beads to the side of an eppendorf tube with a Magnetic Particle Concentrator (Dynal UK) so that the supernatant could be removed. This was repeated three times (All subsequent bead manipulation were carried out in this manner according to manufacture's instructions) .
  • the beads were resuspended in lOOul of TES and the Sau 3A digested DNA added and incubated at room temperature for 1 hour to allow the biotinylated DNA to bind to the beads .
  • the Beads/DNA were then washed three times with Ixligase buffer using the Magnetic Particle Concentrator (Dynal UK) as before.
  • the beads/DNA were was 2 times with 75 ul of lx Fok I buffer and the resuspended in lOOul of lxFok I buffer and heated at 65oC in a Techne Dryblock for 20 minutes to inactivate any remaining ligase.
  • the buffer was removed and the beads/DNA resuspended in 95ul of lx Fok I buffer containing 20 units of Fok I (New England Biolabs)
  • the beads/DNA were then incubated at 37oC for 2 hours. Following incubation the supernatant, containing the fragments cleaved by Fok I, was then transferred to a fresh eppendorf tube and heated at 65oC for 20 minutes in a Techne Dryblock in inactivate the Fok I.
  • Fok I fragments were then divided into three tubes each containing 30ul of Fok I cleaved fragments, 5ul of lOx Ligase buffer, 3ul ligase (at 400uints/ul -New England Biolabs) and 12ul of H20.
  • the ligase buffer on a plate containing adaptors Bbvl4 , 16, 19 in separate wells was removed and the above reaction mixtures, containing the Fok I cleaved fragments and ligase, added to each.
  • the reading obtained from the Bbvl6 well should be half (i.e. 50%) of that obtained from the Bbvl4 well and as one fifth the amount of exon 19 compared to exon 14 (6pmol exon 19, 30 pmol exon 14) the reading obtained from the Bbvl9 well should be one fifth (i.e. 20%) that obtained from the Bbvl4 well.
  • this process is capable of separating a mixed population of DNA , and identifying 4bp, while at the same time maintaining the relative proportions of the original mixture with minimal errors . Which in turn can then be reprobed to obtain another 4bp and the associated quantitative data.
  • the ligation reaction is a critical step in this sequencing technology. Therefore, full optimisation of this reaction is required to ensure success with these techniques.
  • the conditions for the ligation reaction have been investigated by ligating fluorescently (FAM) labelled adaptors to biotinylated adaptors captured to a streptavidin coated microtitre plate.
  • the biotinylated adaptors consist of a GC rich and AT rich type having the 4 base pair overhang sequence CGGC and TAAT respectively. These represent the extremes of GC and AT hybridisation and are therefore used to determine the conditions required to equalise their differing hybridisation kinetics.
  • reaction time increases the amount of FAM labelled adaptor ligated, as expected.
  • a reaction time of 60 minutes will be impractical for the proposed techniques.
  • these reactions do not contain any agents which promote ligation through intra molecular crowding such as polyethylene glycol (PEG) or ficol.
  • PEG polyethylene glycol
  • the intra molecular crowders PEG, ficol and hexamine chloride were titrated to investigate their effects on ligation.
  • Tetremethly ammonium chloride which modifies Watson and Crick base pairing, was also titrated to investigate its effect on the differing efficiency of ligation of AT and GC rich adaptors 5pmol of adaptor was ligated for 10 minutes at 16°C.
  • Figure 9 shows a graph representing the effect that increasing Ficol concentration has on the efficiency of ligating FAM labelled GCCG adaptor (series 1) to captured CGGC target adaptor and FAM labelled ATTA adaptor (series 2) to captured TAAT target adaptor .
  • Ficol has much less of an effect on the efficiency of these reactions as compared to PEG (see below) and therefore will be of less use in helping to equalise the efficiency of ligation between AT and GC rich adaptors .
  • Figure 9 also shows a graph representing the effect that increasing PEG concentration has on the efficiency of ligating FAM labelled ATTA adaptor to captured TAAT target adaptor.
  • Figure 9 also shows a graph representing the effect that increasing PEG concentration has on the efficiency of ligating FAM labelled GCCG adaptor to a captured CGGC target adaptor.
  • the ATCA mismatched adaptor does not ligate to any measurable degree .
  • the presence of the C in the ATCA adaptor must therefore disrupt the base pairing completely thereby preventing any ligation.
  • the ATAA adaptor only ligates at 6.7% of the amount as the ATTA adaptor.
  • the replacement of the T with an A in .this mismatch therefore disrupts base pairing to a lesser degree than a C and therefore allows some ligation.
  • the ligation of this mismatched adaptor is completely displaced by the presence of any unlabelled specific ATTA adaptor.
  • the mismatched adaptors do ligate as compared to the AT rich one which do not .
  • the amount of ligation achieved is reduced to 23% for the GCAG and 10% for the GCGG adaptors.
  • a sequencing reaction by this method involves repeated cycles of cleaving a template with a type IIs restriction endonuclease whose recognition sequence is provided by an adaptor. If the reaction is peformed with multiple templates then each cycle of the sequencing reaction will generate a signal for a series of n-mers. Many cycles will of the reaction will generate a matrix of n-mers which must be analysed to reconstruct the sequences of the source templates .
  • the program operates by first analysing the data matrix to identify in each column of the matrix, corresponding to one cycle of the sequencing reaction, n-mer frequencies or quantities which are equivalent in other columns of the matrix given a predefined margin of error in the measurement of n-mer quantities within which to operate.
  • the raw n-mer frequencies in the data matrix are then replaced with their probable group frequencies in each column .
  • This new data matrix is then analysed by a second algorithm which assumes that there should be the same number of n-mers in each column of the matrix and attempts to resolve any 'sums' of frequencies where the same n-mer has occurred in more than one template in a given cycle of the sequencing reaction.
  • This algorithm takes the group frequencies in the data matrix and generates a sorted 'frequency list' that lists the number of occurrences of each group frequency in order of increasing number of occurrences .
  • the algorithm then takes group frequencies with the lowest number of occurrences first on the assumption that these are likely to be sums, since sums of groups should occur with a relatively low frequency.
  • An alternative would be to generate a sorted list of group frequencies, in order of decreasing quantity, and start with the largest quantities, again on the assumption that these are likely to be sums.
  • the algorithm tests each frequency in the list against each column of the original data matrix. If the group frequency occurs in the column it is tested against all combinations of pairs of group frequencies that are missing from the column to see if any of these missing frequencies can add up to give the current frequency being tested. If any of these missing frequencies do add up and there is only one pair that can add up within the predetermined margin of error then it is assumed that the larger frequency is the sum of the two missing frequencies and the larger frequency is replaced in the current column of the data matrix by occurrences of the two missing frequencies . Any frequencies are the sum of two pairs of missing frequencies are marked as such and in the final sequence reconstruction the bases are marked as unknown.
  • the SeqMatrix data structure stores the matrix of n-mers generated by a sequencing reaction.
  • noise subtraction algorithms would be needed and an algorithm to normalise frequencies in each column to account for progressive decrease in signal with each cycle of the sequencing reaction that will result from the fact that no enzymatic step will be 100% efficient.
  • newList AppendElement(newL ⁇ st, NewElement(meanFreq, groupCount));
  • tempL ⁇ st5 tempL ⁇ st5-> ⁇ ext
  • ⁇ temp ⁇ st4 tempL ⁇ st4->next
  • ⁇ tempL ⁇ st3 tempL ⁇ st3-> ⁇ ext
  • 1 I tempL ⁇ st2 tempL ⁇ st2->next
  • Step 1 Cleave genomic DNA with type IIs restriction endonuclease
  • Step 2 Add adaptors to fragments each bearing primer binding sites such that each sticky-end or subset thereof bears a unique primer site
  • Step 3 Differentially amplify by PCR by adding different amounts of primer for each adaptor
  • Step 1 Cleave genomic DNA with type IIs restriction endonuclease
  • Step 2 Ligate adaptor pair to fragments to tag termini
  • Step 3 Capture fragments to allow fragments with adaptor 2 at both termini to be washed away
  • Step 4 Cleave adaptor 2 with restriction endonuclease
  • Step 6 Ligate capture adaptor to blunt end generated from fragments with adaptor 2 at one end
  • Step 7 Capture fragments or perform arbitrary further sorting - 73b
  • Sorting step Sort fragments onto array of oligonucleotides or into array of 256 wells
  • Cleavage step Cleave immobilised fragments with type IIs restriction endonuclease corresponding to directionality adaptor 1

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Abstract

Procédé pour séquencer de l'acide nucléique, consistant à: (a) obtenir une population cible d'acide nucléique composée de fragments d'acide nucléique et dans laquelle chaque fragment est présent en nombre unique et présente à un bout une séquence d'extrémité cohésive de longueur prédéterminée et de séquence inconnue, (b) protéger l'autre extrémité de chaque fragment, et (c) séquencer chacun des fragments (i) en mettant les fragments en contact avec un ensemble d'oligonucléotides adaptateurs dans des conditions d'hybridation, chaque oligonucléotide adaptateur portant un marqueur, un site de reconnaissance de l'enzyme de séquençage, et une séquence nucléotidique unique connue qui a la même longueur prédéterminée que la séquence d'extrémité cohésive, l'ensemble contenant toutes les séquences de base de cette longueur prédéterminée; en retirant tout oligonucléotide adaptateur non hybridé et en relevant la quantité de tout oligonucléotide adaptateur hybridé par détection du marqueur, puis en répétant toute la procédure, jusqu'à ce que tous les adaptateurs de l'ensemble aient été testés; (ii) en mettant en contact tous les oligonucléotides adaptateurs hybridés avec une enzyme de séquençage qui se lie au site de reconnaissance et coupe le fragment de manière à exposer une nouvelle séquence d'extrémité cohésive, contiguë ou partiellement superposée à la séquence d'extrémité cohésive précédente; (iii) en répétant les étapes (i) et (ii) un nombre suffisant de fois et en déterminant la séquence du fragment en comparant les quantités relevées pour chaque séquence d'extrémité cohésive.
PCT/GB1997/002734 1996-10-04 1997-10-06 Sequençage d'acide nucleique par ligature d'adapteurs WO1998015652A1 (fr)

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