WO2002099129A2 - Mise au point d'amorces pcr degenerees - Google Patents

Mise au point d'amorces pcr degenerees Download PDF

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WO2002099129A2
WO2002099129A2 PCT/GB2002/002640 GB0202640W WO02099129A2 WO 2002099129 A2 WO2002099129 A2 WO 2002099129A2 GB 0202640 W GB0202640 W GB 0202640W WO 02099129 A2 WO02099129 A2 WO 02099129A2
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primer
pcr
primer pair
sequences
sequence
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PCT/GB2002/002640
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WO2002099129A3 (fr
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David John Griffiths
Paul Kellam
Robert Anthony Weiss
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University College London
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Publication of WO2002099129A3 publication Critical patent/WO2002099129A3/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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the invention relates to a method of designing a panel of primers for detecting viruses in a high throughput polymerase chain reaction assay.
  • Viruses require the use of the cellular- translation and transcription machinery to replicate. In the process of replication they often have deleterious effects on the host cell and thus on the host organism. Viruses constitute an important class of pathogens causing many diseases, leading to loss of life in humans and economic loss in the agricultural industries.
  • the polymerase chain reaction allows the amplification of a specific region of a polynucleotide.
  • the specificity of the reaction is due to the primers which during the course of PCR bind to the region to be amplified in a sequence specific manner.
  • the invention provides a method of designing primers which can be used in high throughput screening to detect viruses. The method may be used to detect unknown viruses which have not yet been sequenced.
  • the invention provides a method of designing a panel of degenerate primer pairs for screening for new members of multiple known virus families in a biological sample, wherein each primer pair in the panel binds a sequence that is conserved across members of a said virus family and selectively directs amplification of sequence of said family by PCR, which method comprises
  • each primer in the pair binds a nucleotide sequence that encodes a conserved region identified in (b) and wherein the primer pair is designed to amplify by PCR the nucleotide sequence between the nucleotide sequences that encode conserved regions in members of the first virus family, and
  • the invention also provides a method of designing a panel of degenerate primer pairs for screening for new members of multiple known virus families in a biological sample, wherein each primer pair in the panel binds a sequence that is conserved across members of a said virus family and selectively directs amplification of sequence of said family by PCR, which method comprises (a) providing a plurality of nucleotide sequences from members of a first virus family,
  • each primer in the pair binds a conserved region identified in (b) and wherein the primer pair is designed to amplify by PCR the nucleotide sequence between the conserved regions in members of the first virus family, and
  • the invention additionally provides a panel of primers which has been designed by the method of the invention.
  • the invention provides a method of designing a panel of primer pairs which can be used in high throughput virus screening.
  • the method comprises initial steps which deduce the sequences of the primers using computer based calculations, and optional later steps in which the primers are synthesised and tested empirically, for example to determine optimal PCR conditions and/or to select primer pairs with desired further properties.
  • the panel of primers provided by the method are designed to be capable of detecting unknown viruses based on nucleotide and/or amino acid sequences in the unknown virus which are similar (homologous) to nucleotide and/or amino acid sequences in a known virus. These conserved sequences typically have a role in providing a necessary or advantageous activity or property to the virus. conserveed nucleotide sequences may be coding or non-coding sequences.
  • the panel of primers is designed to detect viruses which are single stranded or double stranded DNA or single stranded or double stranded RNA viruses.
  • the viruses are generally capable of infecting prokaryotic or eukaryotic cells, such as bacterial, animal, plant, yeast or fungal cells.
  • the viruses are mammalian (preferably primate) or avian viruses, such as human, pig, horse, sheep, goat, cow, chicken, turkey or duck viruses.
  • the viruses are typically from any combination of the following families: Adenoviridae, Arenaviridae, Arteriviridae, Astroviridae, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Deltavirus, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Polydnaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Togaviridae or Bornavirus.
  • the primers of the panel are capable of detecting unknown viruses in a biological sample. Such a sample either originates from a single individual or is a pooled sample from individuals of the same species. Thus the panel of primers detects viruses which infect the same species (from which the sample originates).
  • primer pairs designed in the method bind sequence which is conserved across members of a virus family.
  • the panel which is designed in the method may comprise primer pairs that bind sequence which is conserved across substantially all members of the family or across a subset of the members of the family, for example across all members of a subfamily or of a genus.
  • the primer pairs bind at least 70%, at least 80%, or at least 90% of the known viruses of the family, subfamily or genus. Preferably less than 10, such as less than 5, primer pairs will be used for the detection of any given family, subfamily or genus in the panel.
  • the panel of primer pairs is generally capable of detecting viruses from at least 10, 15, 20, 30 or more families, typically up to a maximum of 35 families.
  • the panel of primer pairs may comprise sets of primer pairs which perform a nested PCR reaction.
  • a set of primer pairs comprises a first and second primer pair.
  • the first primer pair is able to amplify a template nucleotide sequence from a virus to form a PCR product.
  • the second primer pair is able to amplify a nucleotide sequence using the PCR product generated by the first primer pair as a template.
  • the use of nested sets of primer pairs allows increased sensitivity.
  • each primer pair is specific for a particular virus family, so that it does not detect viruses of other families .
  • the plurality of amino acid sequences or nucleotide sequences are provided from different known viruses of the same family.
  • the amino acid sequences or nucleotide sequences will be for the same protein of the different viruses. Typically at least 5, 10, 20, 50, 100 or more sequences are provided. The maximum number of sequences provided will, for example, be 300 sequences.
  • Each of the sequences which is provided is typically at least 20, 50, 100, 200 or more amino acids or nucleotides in length.
  • the maximum length of the nucleotide sequences is 1000 nucleotides and the maximum length of the amino acid sequences is 300 amino acids.
  • the sequences may be obtained from a database of sequences, such as GenBank.
  • the sequences may be obtained from a database comprising virus sequences which are organised into homologous protein families (based on sequence similarity relationships).
  • sequences are obtained from the VTDA database (described in Alba et al (2001) Nucleic Acids Research 29, 133-136) or the Virus Division of GenBank.
  • the sequences may be provided in the form of a database, preferably in computer-readable form.
  • the sequences are preferably provided in the form of a computer-readable database constructed using programs which identify homologous protein families, such as GeneTableMaker, MKDOM or PSCBuilder.
  • conserved regions typically such conserved regions will have a length of at least 12 nucleotides, such as at least 15, 21, 27, 36, 99 or more nucleotides (generally up to a maximum length of 200 nucleotides) or at least 4, 5, 7, 10, 25 or more amino acids (generally up to a maximum length of 50 amino acids).
  • the virus sequences which are being provided will of course share identity or similarity.
  • the amino acids or nucleotides in at least 50% of the positions in the region will be the same in at least 50 %, 60%, 70%, or 80% of the viruses of the group (i.e. in the family, genus or subfamily).
  • the algorithm which identifies conserved regions generally uses a multiple sequence alignment method.
  • the method may comprise (a) aligning all pairs of sequences separately to calculate a distance matrix giving the divergence of each pair of sequences, (b) calculating a guide tree from the distance matrix, and (c) aligning the sequences progressively according to the branching order in the guide tree.
  • a preferred algorithm for the aligning the conserved sequences is CLUSTALW as described in Thompson et al (1994) Nucleic Acids Research 22, 4673-80.
  • Other algorithms that can be used for aligning sequences are MultAlin (Corpet (1988) Nucleic Acids Research 16, 10881-90) or Jalview (Clamp et al (1998) http://barton.ebi.co.uk).
  • BLOCKS of conserved regions of amino acids may be extracted from the multiple alignments, typically using the program Blocks Multiple Alignment Processor. Alternatively the entire process of performing multiple alignments and extracting BLOCKS can be performed using BLOCKMAKER (Henikoff and Henikoff (1994) Genomics 19, 97-107).
  • the output from the alignment and BLOCK extraction set i.e. the information describing the identified conserved regions
  • Such output is typically in the form of partial sequences which correspond to the conserved regions (BLOCKS).
  • BLOCKS are input into a primer design algorithm. In one embodiment such an algorithm is CODEHOP.
  • the conserved regions which are chosen as targets for primers preferably comprise few codohs with degenerate counterparts, i.e. preferably the sequence has a low redundancy, such as a redundancy of less than 512 fold, 256 fold or 128 fold.
  • Each primer binds in accordance with Watson-Crick base pairing and thus the binding is sequence specific.
  • Each primer will thus be designed to be wholly or partially complementary to the sequence to which it binds.
  • Each of the primers typically has a length of at least 8 nucleotides, such as at least 10, 12, 15, 20, 30, 40 or more nucleotides (up to a maximum of 50 nucleotides for example).
  • the primer may. comprises at least 2, 4 or 6, up to a maximum of 10 for example, inosine bases. Inosine is able to bind to any of the four nucleotides and therefore use of inosine causes a reduction in effective redundancy.
  • Each primer pair will be designed so that the PCR product generally has a length of at least 20, such as at least 50, 100, 200, 500, 1000 or more nucleotides (and typically up to a maximum of 5xl0 3 nucleotides long).
  • Each primer is preferably designed so that it anneals to a single site, i.e. the primer will not bind to any other site in the genome of the relevant virus.
  • Each primer is preferably designed so that it does not exhibit secondary structure, i.e. the nucleotides in the primer will not bind substantially to any other nucleotide in the primer apart from those to which it is covalently linked.
  • each primer is designed so that it does not bind other primers with the same sequence.
  • the 3' region, and preferably the 3' terminal nucleotide of the primer binds to the target sequence with high affinity, thus preferably this region or nucleotide comprises a G or C.
  • each primer is designed to have an annealing temperature of from 30 to 65 ° C, such as 50 to 60 ° C or 35 to 45 ° C.
  • each primer pair may be designed to ensure that the two primers do not bind to each other.
  • such an algorithm designs primers according to the following rules:
  • a set of blocks is input, where a block is an aligned array of amino acid sequence segments without gaps that represents a highly conserved region of homologous proteins.
  • a weight is provided for each sequence segment, which can be increased to favour the contribution of selected sequences in designing the primer.
  • a codon usage table is chosen for the target genome.
  • PSSM amino acid position-specific scoring matrix
  • step 4 For each position of the block, the most common codon corresponding to the amino acid chosen in step 3 is selected utilizing the user-selected codon usage table. This selection is used for the default 5' consensus clamp in step 8. 5)
  • a DNA PSSM is calculated from the amino acid matrix (step 2) and the codon usage table.
  • the DNA matrix has three positions for each position of the amino acid matrix.
  • the score for each amino acid is divided among its codons in proportion to their relative weights from the codon usage table, and the scores for each of the four different nucleotides are combined in each DNA matrix position. Nucleotide positions are treated independently when the scores are combined. As an option, the highest scoring nucleotide residue from each position can replace the most common codons from step 4 that are used in the consensus clamp.
  • the degeneracy is determined at each position of the DNA matrix based on the number of bases found there.
  • a weight threshold can be specified such that bases that contribute less than a minimum weight are ignored in determining degeneracy.
  • Possible degenerate core regions are identified by scanning the DNA matrix in the 3' to 5' direction.
  • a core region must start on an invariant 3' nucleotide position, have length of 11 or 12 positions ending on a codon boundary, and have a maximum degeneracy of 128 (this is the default setting of CODEHOP).
  • the degeneracy of a region is the product of the number of possible bases in each position.
  • Candidate degenerate core regions are extended by addition of a 5' consensus clamp from step 4 or 5.
  • the length of the clamp is controlled by a melting point temperature calculation (the CODEHOP default is 60 °C) and is usually about 20 nucleotides. 9) Steps 7 and 8 are repeated on the reverse complement of the DNA matrix from step 5 for primers corresponding to the opposite DNA strand.
  • CODEHOP Rose et al (1998) Nucleic Acids Research 26, 1628- 1635) is used to design the primer pairs. This program uses the above rules.
  • the primers designed by the algorithm may then be mapped back to the original sequence to choose primer pairs which provide the desired length of PCR product.
  • primer pairs can then be synthesised and tested. They are typically tested to determine the optimal conditions for using the primers in a PCR reaction.
  • the PCR reaction is carried out in a PCR mixture that generally comprises the following: the template polynucleotide (which will be amplified in the event of virus detection), one or more primer pairs designed as described above, a polymerase enzyme (typically a DNA polymerase, such as Taq polymerase), deoxynucleotide triphosphates (dATP, dTTP, dCTP and dGTP) and a suitable buffer.
  • a polymerase enzyme typically a DNA polymerase, such as Taq polymerase
  • deoxynucleotide triphosphates dATP, dTTP, dCTP and dGTP
  • the PCR reaction generally comprises cycles of the following steps: a denaturation step, a primer annealing step and a polynucleotide synthesis step.
  • the PCR reaction comprises at least 25 cycles, such as 30, 35, 40 or more cycles, up to a maximum of 60 cycles for example.
  • the PCR mixture is heated to a temperature at which the polynucleotides in the PCR mixture (in particular the polynucleotide region to be amplified) denature to single stranded form.
  • the denaturing temperature is generally from 85 to 98 "C.
  • the primers bind to template nucleotide sequence in a sequence specific manner.
  • This step is generally carried out at a temperature of from 30 to 65 °C.
  • the polymerase replicates/synthesises nucleotide sequence based on template sequence by addition of nucleotides to the 3' end of the bound primers. This step is generally carried out at about 72 ⁇ 0
  • the primers are tested for their ability to amplify one or more of the plurality of nucleotide sequences from known viruses which were used to design the primers, or in the case of amino acid sequences from known viruses being used to design the primers the primers may be tested for their ability to amplify the nucleotide sequence from the virus which encodes the amino acid sequence.
  • the primers may be tested in a range of buffer conditions to determine optimal buffer conditions for PCR using the primers.
  • the buffer conditions which may be tested include pH (typically between 7 and 10), magnesium concentration (typically from 0.5 M to 5 mM), potassium chloride (typically from 0 to 100 mM), ammonium chloride (typically 0 to 100 mM), glycerol (typically 0 to 20%), dimethysulphoxide (typically 0 to 20%), ethanol (typically 0 to 20%), sorbitol (typically 0 to 20%) or betaine (typically 1M betaine).
  • pH typically between 7 and 10
  • magnesium concentration typically from 0.5 M to 5 mM
  • potassium chloride typically from 0 to 100 mM
  • ammonium chloride typically 0 to 100 mM
  • glycerol typically 0 to 20%
  • dimethysulphoxide typically 0 to 20%
  • ethanol typically 0 to 20%
  • sorbitol typically 0 to 20%
  • betaine typically 1M betaine
  • the primers may be tested at a range of different temperatures to determine the optimal temperatures in the PCR reaction.
  • the primers are tested in PCR reaction in which a range of primer annealing temperatures are tested.
  • the range of temperatures is from 30 to 65 ° C .
  • the panel of primer pairs or a group of primers within the panel may be designed to be used together on the same plate (i.e. using the same thermal cycles). Thus such primer pairs will be designed to work at the same annealing temperature.
  • a group of primer pairs within the panel are designed to have similar optimal conditions for use in PCR so that they can be used optimally in the same well or reaction vessel, i.e. that they can be used in multiplex PCR.
  • Such a group typically comprises at least 2, 3, 4, 5, 6 or more primer pairs (up to a maximum of 8 primer pairs for example).
  • the computer based method steps may be used to design primer pairs which are calculated to have similar annealing temperatures and/or the primers are tested to select primer pairs which can be used optimally together. Such testing typically determines whether the primers work optimally with the same buffers and/or whether the primers have similar annealing temperatures.
  • At least one or both primers of each primer pair in the group carries a label. Typically at least one of the primers in each primer pair will carry a different label from that used for the other primer pairs.
  • the PCR product generated by labelled primers carries the labels present on the primers. Thus after the group of primers have been used for PCR in the same well detection of the labels in the PCR products can be used to deduce which PCR product was formed from each primer pair.
  • all forward primers of the group are labelled with one colour and the reverse primers are labelled with a different colour.
  • the primers are labelled with a fluorescent label, such as fluorescein based labels (e.g. fluorescein isothiocyanate).
  • a fluorescent label such as fluorescein based labels (e.g. fluorescein isothiocyanate).
  • fluorescein based labels e.g. fluorescein isothiocyanate
  • Different primer pairs may be labelled with fluorescent labels of different colours.
  • the fluorescent labels which are used may be capable of detection by a Beckman CEQ2000TM or Applied Biosystems A3700TM fluorescent DNA analyser.
  • the fluorescent labels may obtained from Beckman Coulter or Applied Biosystems.
  • each PCR product which is generated by the group of primers differs in size from all the other PCR products by at least 20, such as at least 50, 100, 200, 500, 1000 or more nucleotides.
  • Each PCR product may for example differ in size from all other PCR products by up to a maximum of 3000 nucleotides.
  • Example 1 illustrates the invention.
  • a panel of primers was designed for detecting unknown viruses from the family Herpesviridae according to the strategy shown in Figure 1.
  • the amino acid sequences of herpes virus DNA packaging protein UL15 were obtained from the VTDA database (Alba et al, see above). These sequences are shown in Table 1. The sequences obtained from the VTDA database were then imported into
  • CLUSTALW This compares the protein sequences to identify conserved regions and then aligns the sequences according to the conserved regions.
  • the alignment produced by CLUSTALW is shown in Table 2.
  • the BLOCKMAKER program was then used to extract blocks of conserved aligned sequences which do not contain gaps from CLUSTALW and enter them into CODEHOP.
  • the primer sequences were then designed by CODEHOP using the conserved sequences.
  • the output from the CODEHOP program is shown in Table 3.
  • the 'Complement of Block' sequences shown in Table 3 shows the sequence of the other strand allowing primers to be designed for amplification in the opposite direction.
  • RVKAHTNATSCSCYIIJNKPVFITI DGAMRNTAELF PDSFMQEI IGGGNISGAHRDEPVFTKTAQDRF Ii

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Abstract

L'invention concerne un procédé de mise au point d'amorces PCR pour le criblage de nouveaux membres de familles de virus connues.
PCT/GB2002/002640 2001-06-07 2002-06-07 Mise au point d'amorces pcr degenerees WO2002099129A2 (fr)

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