WO2002036815A2

WO2002036815A2 - Genetic analysis of microorganisms

Info

Publication number: WO2002036815A2
Application number: PCT/GB2001/004760
Authority: WO
Inventors: Stephen Minter; James Ivor Prosser; Carol Jane Phillips
Original assignee: Ncimb Ltd.
Priority date: 2000-10-28
Filing date: 2001-10-29
Publication date: 2002-05-10
Also published as: GB0026424D0; WO2002036815A3; GB2390159A; GB0314840D0; AU2002214126A1

Abstract

A method for genetic analysis of a microbial sample by linking taxonomic to functional characteristics comprises co-amplification of a taxonomic gene and a functional gene in a microbial sample and linking of the two amplified products. An analysis may then be performed for the linked product.

Description

GENETIC ANALYSIS OF MICROORGANISMS

The present invention relates to methods for the genetic analysis of microorganisms and more particularly to methods to determine if a particular microorganism has a gene that provides the microorganism with the potential to perform a particular function.

Microorganisms and their metabolic products are of considerable commercial value. They are the source of the majority of antibiotics and other therapeutic agents, are important for the biodegradation of pollutants and are the mainstay of traditional and modern biotechnological processes. This commercial value reflects the genetic, metabolic and physiological diversity of microorganisms.

Many micoorganisms that have a functional gene capable of being expressed to produce a useful product have been identified using conventional diversity studies. The traditional approach for the analysis of microorganism diversity involves identification of pure cultures (isolated on laboratory media) obtained from environmental samples. There is however strong evidence that this approach detects only a small proportion (less than 1%) of the microorganisms present in the original sample due to the selectivity of growth media and conditions.

Analysis of DNA extracted from environmental samples has allowed workers to investigate bacterial communities without cell extraction followed by laboratory cultivation and culture. Broad-scale techniques, such as DNA reassociation and reannealing, provide a measure of total microbial diversity and have revealed the influence of environmental parameters such as pollution and agricultural exploitation on microbial diversity.

More detailed analysis can be performed by using 16S ribosomal DNA (rDNA)-based techniques and a range of methods targeting both rRNA and rDNA are now used routinely in microbial ecology (see Embley, T.M. & Stackebrandt, E. (1996) p 39-62 in W. Pickup and J.R. Saunders (ed.), Molecular Approaches in Environmental Microbiology, Ellis-Horwood, London).

These 16S rDNA-based methods include detection by in situ hybridisation and PCR amplification followed by sequence analysis and denaturing gradient gel electrophoresis. Primers and probes having different specificities, ranging from universal to species-specific, can be used with combinations of these techniques to provide a comprehensive understanding of bacterial community structure.

As well as analysing bacterial diversity, obtaining information on the genetic capabilities and phylogenetic affinities of individual microorganisms within natural communities is a high priority. With few exceptions, it has been impossible to determine which particular strains in a natural microbial sample possess the genetic capability to carry out a specific process and whether or not the gene is being expressed.

Even micro-scale sampling techniques based on viable cell counts offer little promise for providing insight into microbial community structure since the cultivation efficiency of microbes from natural communities is generally very low. A method termed "in situ PCR" (see Hodson, R.E. et al, 1995, Applied and Environmental Microbiology, 61, p 4074-82) allows detection of genes in a single cell and in cultured mixtures of bacterial isolates and model communities. In this method a known microbe is tested to determine if it has a gene that will allow the microbe to perform a particular function. This method involves amplification of specific nucleic acid sequences inside intact prokaryotic cells, followed by colour or fluorescence detection of the localised PCR product via bright-field or epifluorescence microscopy. In situ PCR, as described in Hodson et al, supra., is not appropriate for all prokaryotic species, especially those which are not presently culturable or non- or slow-growing cells, typical of many natural communities.

An example of a group of bacteria having properties of interest which are conferred by functional genes is those bacteria capable of carrying out ammonia oxidation. Extensive phylogenetic studies of ammonia oxidising bacteria have been carried out on a range of environmental samples, including marine and freshwater sediments and bulk waters, cultivated and non-cultivated soils and wastewater treatment sites (McCaig et al, 1994, FEMS Microbiol. Lett., 120 p 363-368; Stephen et al, 1996, Appl. & Environ. Microbiol., 62, p 4147-4154, Kowalchuk et al, 1996, Appl. & Environ. Microbiol., 63, p. 1489-1497, Phillips et al, 1999, Appl. & Environ. Microbiol., 65, p. 779-786, Schramm et al, 1998, Appl. & Environ. Microbiol., 64, p 3480-3485). These studies have utilised 16S rDNA specific primers and oligonucleotide probes and have shown a phylogenetic diversity within this group of organisms that was undetectable using traditional culture based methods. Selection of specific strains of ammonia oxidising bacteria is seen in response to environmental conditions for example sediment pollution (McCaig et al, 1999, Appl. & Environ. Microbiol., 65, p 213-220) and soil pH (Stephen et al, 1998, Appl. & Environ. Microbiol., 64, p 2958-2965). Studies on the phylogenetic diversity of ammonia oxidising bacteria have been paralleled by those on functional gene diversity, by analysis of ammonia monooxygenase (amoX) gene sequences (Rotthauwe et al, supra, Mendum et al, 1999, Appl. & Environ. Microbiol., 65, p 4155-4162). Both molecular approaches demonstrate significant diversity and the existence of organisms and groups of organisms that have never been characterised in laboratory culture. However, it is not possible, using existing technology to link phylogenetic (species) diversity and functional diversity. That is, it is not possible to know which ammonia oxidiser species, determined on the basis of 16S rRNA gene sequence, possesses which amoA gene sequence.

It is an aim of an embodiment of the present invention to provide a method to link distribution and identity of microorganisms with their genetic potential and in situ activities, irrespective of the ability to provide pure or mixed laboratory cultures of the microorganism.

According to a first aspect of the present invention there is provided a method for genetic analysis of a microbial sample by linking taxonomic to functional characteristics, the method comprising co-amplification of a taxonomic gene and a functional gene in a microbial sample and linking and further amplification of the two amplified products.

According to a second aspect of the invention there is provided a method of analysing a microbial sample comprising introducing intracellularly reagents for effecting co- amplification of a taxonomic gene and a functional gene if present in the cell, applying amplification conditions, applying conditions for linking any amplified products of the taxonomic gene with amplified products of the functional gene, and analysing for the linked product.

The linking conditions should be such that any amplified products of the taxonomic gene link only with amplified products of the functional gene.

Preferably, in accordance with the second aspect of the invention, an amplification step is effected to amplify the linked product, if indeed such a product has actually been formed. Analysis may then effected for the amplified linked product. This amplification may be conducted intracellularly or extracellularly.

The method according to the invention allows genetic analysis of a microbial sample to determine which microorganisms are contained in the sample and which of those microorganisms carries a specific functional gene (or other gene of interest) which provides the potential for a particular function. More particularly detection of a linked product comprised of the amplified products of a taxonomic gene and a functional gene provides taxonomic information about the microorganism having that functional gene. This taxonomic information may be used, for example, for isolation of the organism.

This is the first time that it has been possible to analyse an environmental sample to determine which microbes present in that sample have a particular function. The method according to the present invention has the advantage that it may be carried out irrespective of the need to culture and purify the microorganisms. As the diversity highlighted and characterised by molecular analysis of natural populations has indicated that uncultured organisms (99-99.99% of the total population) are a vast and untapped genetic resource the present invention is of considerable commercial importance. This applies both to uncultured members of established groups and to the major, novel and uncharacterised microorganisms discovered through molecular analysis.

The inability to cultivate the majority of microbes in natural populations has previously resulted in ignorance of the physiological properties and function of a wealth of microbes. These unculturable microorganisms may contain properties and genes of commercial importance. If one considers the enormous commercial value that has arisen from microbes that have been cultured and then consider that this may represent only 0.1% of the total diversity, the commercial potential of the present invention, which allows genetic analysis of unculturable microorganisms, becomes evident.

The functional gene may be one capable of providing a desired product, it may the one capable of degrading a particular product, or may be one conferring resistance to a particular agent (e.g. antibiotic resistance). Non-limiting examples of functional genes that may be "targetted" using the techniques of the invention are given in the following Table.

The method of the invention may be used for producing linked copies of a taxonomic gene and two or more functional genes.

The method of the invention is preferably carried out on intact cells so that the functional gene identified can be unambiguously linked to a particular cell, which can be identified by its taxonomic genes.

According to a preferred embodiment, the method of the first aspect of the invention is preceded by the steps of:

(a) extraction of cells from an environmental sample;

(b) immobilisation of intact extracted cells on a solid support; and

(c) semi-permeabilisation of the immobilised cells to enable entry of reagents (including for example primers and other reagents) for amplification. The microbial sample may be for example a culture of one or more microbes or an enrichment culture. However, the present invention finds particular utility when the microbial sample is a natural microbial sample, for example an environmental sample such as a soil, water or oil sample, especially when such a sample contains unculturable microbes.

The microbial sample for analysis may be obtained by extraction of cells from the environmental sample using a variety of established techniques such as sonication, washes with detergents such as Tween, bead beating in the presence of a saline solution and dispersion in a Waring blender with sodium phosphate buffer.

The extracted cells may be immobilised on a solid support by a variety of techniques, for example by filtration. According to a preferred embodiment the isolated microbial cells are immobilised on beads such as paramagnetic beads, the beads washed and placed in a well or matrix, under serial dilution, preferably to provide one cell per well/matrix.

According to a preferred embodiment of the invention to immobilise a cell on a bead, beads of approximately 100 μm in diameter are added to a sample of microbes in a container. Each bead is designed to pick up an individual microbial cell, for example by the bead being coated with a binding partner specific for an agent on the surface of the cell, such as an antibody to the cell, or by passive adsorption using "sticky" beads. Beads that have specificity for a particular type or classification of microbe could be used, for example which carry on their surface 16S rDNA for a particular type of microbe, so that for example, only proteobacteria attach to the beads. The use of antibody bearing beads or 16S rDNA bearing beads increases the selectivity and specificity of the method

The immobilised cells are then washed and each bead placed in a well/matrix by using a dilution series. This stage of the procedure is particularly adaptable to automation. If more than one cell is provided per well a mixed product of amplification may be provided. If two cells are provided then there could potentially be four amplification products. In this situation it is advisable to screen the products or to provide a further dilution series and repeat the amplification steps.

Semi-permeabilisation of the cells to enable entry of amplification reagents is preferably carried out using a method such as that described in Hodson et al, supra. In this method cell wall permeabilisation is achieved by treatment of the cells with lysozyme at a final concentration of 0.5 mg/ml for 30 min at room temperature, with removal of lysozyme from the cells by washing with phosphate buffered saline (PBS). Permeabilisation may be furthered by treatment with proteinase K or trypsinogen.

The primers for the taxonomic gene are preferably 16S rDNA primers, such as for example described in Embley et al, supra. The 16S rDNA primers may be for all microbes, for a specific class of microbe, or for a specific strain of a class of microbe. In this way all of the microbes present in a sample, all of the strains of a particular class of microbe that are present in a sample or a specific strain of a class of microbe present in a sample may be analysed.

The primers for the functional gene may be specific for a gene of interest, for example the nahA gene as described in Hodson et al, supra, or the amoA gene of nitrifying bacteria as described in Rotthauwe, J.-H. et al, 1997, Applied and Environmental Microbiology 63, p 4704-4712. Alternatively, the functional gene primers may be random primers that may be used to amplify previously unknown genes, which may be further analysed by functional genomics and comparison with genetic databases.

Amplification is preferably carried out using standard polymerase chain reaction (PCR) techniques. In situ PCR is particularly applicable to the method of the present invention.

The linking of the amplified products may be effected by means of a linker molecule.

According to a preferred embodiment, the method of the first aspect of the invention compnses:

(a) providing a microbial sample with primers specific for a taxonomic gene and a functional gene;

(b) carrying out an amplification reaction to provide a taxonomic gene fragment and a functional gene fragment;

(c) adding a linker molecule to create a hybrid of the taxonomic gene fragment and functional gene fragment;

(d) co-amplification of the hybrid product; and

(e) detection and analysis of the amplified hybrid product.

Previously it has been possible to characterise taxonomic genes and functional genes separately. The use of a linker molecule to create a hybrid according to the first aspect of the invention allows the taxonomic gene to be linked to the functional gene to show, for the first time, which microbes in a sample comprise genes for performing a particular function.

The linker molecule preferably has one end that is complementary to a recognition site in the taxonomic gene primer and another end that is complementary to a recognition site in the functional gene primer.

Addition of the linker molecule to the amplified taxonomic gene fragment and functional gene fragment is preferably done in the presence of lambda exonuclease and Klenow fragment (3 '-5'). The lambda exonuclease promotes the formation of "sticky ends" (3' overhangs) on the linker fragment and amplified fragments and the Klenow fragment fills in 3' overhangs to produce a hybrid product made up of the taxonomic gene fragment joined to the linker joined to the functional gene product. The mechanism behind this linking is described in Hung Tseng, 1999, BioTechniques 27, p 1240-1244 in relation to DNA cloning without using restriction enzymes or ligase.

The hybrid product may be subjected to amplification with one primer specific for the taxonomic gene fragment and one primer specific for the functional gene product, so as to produce an amplification product containing the two genes linked (but separated by the linker molecule). The amplified hybrid may be sequenced to determine which functional genes are associated with which taxonomic genes and therefore which microbes in a sample contain genes that allow them to carry out a particular function.

An alternative embodiment of the invention comprises:

(a) providing isolated microbial cells on a solid phase in a well or matrix, the solid phase carrying known sequences x and y;

(b) providing primers for a taxonomic gene fragment and a functional gene fragment, the primers carrying extension sequences x' and y', which are capable of hybridising to sequences x and y on the solid support;

(c) carrying out an amplification reaction to provide taxonomic and functional gene products carrying extension sequences x' and y';

(d) linking the amplification products at x' and y' to provide a hybrid product comprising the taxonomic gene fragment, x', y' and the functional gene product;

(e) amplifying the hybrid product.

The use of sequences x, y, x' and y' allows the hybrid product to be "fished out" of the well and supported on the solid phase. This means that the starting material in the well or matrix need not be one or two cells per well as is preferred in relation to the earlier embodiment.

The primers used in accordance with step (b) of the alternative embodiment are preferably the same as described in relation to the earlier embodiment. Amplification is preferably carried out by PCR.

Linkage of x' to y' may be carried out using DNA ligase or may be performed as described in relation to the earlier embodiment. As with the earlier embodiment the amplified hybrid may be sequenced to determine which functional genes are associated with which taxonomic genes and therefore which microbes in a sample contain genes that allow them to carry out a particular function.

According to a further embodiment of the first aspect of the invention linkage may be achieved by the production of amplification products of the taxonomic gene which contain a "tail" which is of a sequence complementary to that of a further "tail" provided on an amplification product of the functional gene. In such a case the production of a linked product comprising the amplified taxonomic gene fragment and the amplified functional gene fragment is brought about by the hybridisation of the complementary "tails" provided on the products. It is preferred that the amplified taxonomic gene fragment and the amplified functional gene fragment be provided with different complementary tails in order that hybridisation of fragments from the same gene may be avoided.

The tails used in the method of linkage outlined above may, for example, be sequences of DNA complementary for one another, but not for the genes of interest, provided as parts of the primers used for the amplification.

The linked product produced by hybridisation of amplified gene products having tails may be further amplified prior to sequencing. Such further amplification may be performed using either intracellular or extracellular methods as desired. Use of intracellular amplification provides an advantage in cases where the technique is applied to mixed populations of cells since it allows greater certainty that both the taxonomic and functional genes are found within the same cell.

The present invention will now be described by way of example only with reference to the following non-limiting Examples and accompanying drawings in which:

Figure 1 illustrates the steps that may be involved in co-amplification of two remote genes for comparison of sequence variability in a taxonomic gene with that of the functional gene wherein linkage of the amplified products is achieved using a linker molecule;

Figure 2 illustrates the amplification and linkage of a taxonomic gene fragment and a functional gene fragment wherein linkage of the amplified gene fragments is achieved through the hybridisation of mutually complementary regions of DNA; and

Figure 3 illustrates the result of Example 3.

Figure 1 (a) shows a section of genomic DNA from a microbial sample, showing a taxonomic gene remote from a functional gene. Numbers 1 and 2 represent 16S rDNA taxonomic gene specific primers, which could be either general or specific, and numbers 3 and 4 represent primers for the functional gene, which may be specific to a gene of interest or may be random.

PCR according to Figure 1(a) produces two PCR products, A and B, shown in Figure 1(b). Figure 1(b) also shows a linker molecule that has ends, that are complementary to PCR primer recognition sites 2 and 3 in Figure 1(a).

Figure 1(c) shows the result of linker fragment and PCR product digestion with lambda exonuclease to produce sticky ends, with subsequent hybridisation of the ends of the linker molecule with primer recognition sites 2 and 3.

Figure 1(d) shows the repair of single stranded breaks by Klenow fragment (3 '-5').

Figure 1(e) shows the hybrid product produced by PCR with primers 1 and 4. This hybrid product comprises a taxonomic gene fragment and a functional gene fragment linked by a linker molecule (of known sequence). Sequencing and analysis of the hybrid product allows determination of which functional genes are associated with which taxonomic genes and therefore which microbes in a sample contain genes that allow them to carry out a particular function. Turning now to Figure 2, Figure 2(a) shows a taxonomic gene 5 remotely situated from a functional gene 6. Paired PCR primers 7 and 8 are specific for the taxonomic gene 5, whilst paired primers 9 and 10 are specific for the functional gene 6. In addition to sequences capable of specific binding to their target genes primers 8 and 10 further comprise "tail" portions (8' and 10'), the tail portions of primer 8 and primer 10 being complementary to one another.

Figure 2(b) shows an amplification product 11 of gene 5 using primer pair 7 and 8 , product 11 comprising DNA strands 12 and 13. Also shown is an amplification product 14 of gene 6 using primer pair 9 and 10 , product 14 comprising DNA strands

15 and 16. Products 11 and 14 are both comprised of double stranded DNA substantially derived from the amplified gene fragments. However, since DNA strand 13 of product 11 incorporates primer 8, and hence the tail portion 8', so DNA strand 12 contains a portion of DNA 12' complementary to the tail 8' sequence. Similarly as DNA strand 15 of product 12 incorporates primer 10, and so tail 10', DNA strand 16 contains a sequence 16' complementary to the sequence of tail portion 10'.

Figure 2(c) shows that upon denaturating of products 11 and 14 DNA strands 12 and

16 are able to hybridise to one another through the sequence portions 12' and 16' complementary to the tail sequences. DNA polymerase is then able to initiate polymerisation from this hybridised region to produce strands complementary to the gene-derived sequences of DNA strands 13 and 16 (as illustrated by the broken arrows).

Thus a double stranded fusion product of fragments of genes 5 and 6, linked by sequences corresponding to tail portions 8' and 10' and regions 12' and 16', is produced, as shown in Figure 2(d), in which the fusion product is generally designated 17.

Figure 2(e) illustrates the amplification of fusion product 17 using primers 7 and 9. Such an amplification may be performed to facilitate analysis of the product 17.. EXAMPLE 1

In-situ linking and amplification of taxonomic and functional genes in C. albicans cells

A first broth culture of C. albicans was set up by inoculating 10 ml of YPD broth with a single C. albicans colony. The broth was incubated at 30°C with shaking overnight. To ensure that the cells to be used were in the exponential phase of growth, during which they are most active, 500 μl of the first broth culture was taken to inoculate 10 ml of fresh YPD medium and incubated at 30°C for a further 3 h to establish a second broth culture.

The cells of this second broth culture were then pelleted by centrifugation at 5,000 rpm for 5 min. The cells were washed three times by re-suspending the cells in 1 ml PBS by pipetting and then centrifuging the cells at 5,000 rpm for 2 min.

After the final wash the cells were re-suspended in 460 μl of buffer A [0.1 M potassium phosphate buffer (pH 7.5), 1.2 M sorbitol]. An aliquot of these cells was retained for use as a control (representing non-permeabilised cells). The remaining cells were permeabilised by incubation with 40 μl zymolyase and 1 μl β- mercaptoethanol at 37°C for 15 min with gentle agitation. The cells were then washed again three times with PBS as above. The final cell pellet was resuspended in 100 μl of PBS.

The taxonomic and functional genes selected for amplification and hybridisation were the 18S rRNA gene and the chitin synthase gene respectively. The primers pairs used to achieve amplification and linking of the genes were nu-SSU-0817 and nu-SSU- 1536TAIL, which are specific for the 18S rRNA gene, and CHS8-8TAIL and CHS8- 11, which are specific for the chitin synthase gene (details of the primers are included in Table 1). nu-SSU-1536TAIL and CHS8-8TAIL both contain, in addition to sequences complementary to their respective target genes, "tail" sequences that complement one another. In situ PCR reactions were set up such that each 50 μl reaction volume contained:

2.5 U of Taq DNA polymerase (Bioline, UK);

10X reaction buffer [160 mM (NH₄)₂SO₄, 670 mM Tris-HCl (pH 8.8 at 25°C), 0.1%

Tween-20];

250 μM dNTPs;

20 pmol of nu-SSU-0817 and CHS8-11 primers; and

0.2 pmol of nu-SSU-1536TAlL and CHS8-8TAIL primers.

PCR amplifications were carried out on a Hybaid Omni-E Thermal Cycler (Hybaid) as follows: 94°C for 5 min (one cycle); 94°C for 40 s, 55°C for 2 min, 74°C for 1 min (10 cycles); 94°C for 40 s, 55°C for 40 s, 74°C for 1 min (23 cycles) followed by a final incubation at 74°C for 7 min.

The presence of relatively larger quantities of the "external" primers (nu-SSU-0817 and CHS8-11) than of the TAIL primers (nu-SSU-1536TAIL and CHS8-8TAIL) favours amplification of the hybridised product.

Following completion of the PCR, the cells were pelleted by centrifugation at 5,000 rpm for 2 min. The supernatant was removed and retained for analysis. The cells were then washed three times with 200 μl PBS as described above. Following the third centrifugation, the supernatant wash solution was retained for analysis. The final cell pellet was resuspended in 30 μl PBS and incubated at 95°C for 5 min to lyse the cells. The original supernatant from the PCR, the wash solution, and the lysed cell solution were then analysed by electrophoresis in a 1% (w/v) ethidium bromide stained agarose gel.

Visualisation of the PCR reaction products present in the supernatant from the in situ PCR reaction mixture is shown in Figure 3. Reaction products are present in lanes 1 to 6. The reaction products contain the hybridisation product, comprising fragments of the 18S rRNA gene and the chitin synthase gene linked by the hybridised "tail" sequences (band indicated with arrow), 18S rRNA product and chitin synthase product. Excision and sequence analysis of the hybrid product band has confirmed its identity through the presence of fragments of both target genes. The lane labelled M contains molecular weight markers, and the lane labelled C represents a negative control.

Table 1. PCR primers (complementary tail sequences shown in bold).

EXAMPLE 2

Amplification of dioxygenase genes from Arthrobacter sp cultures in the laboratory.

A 10 microlitre aliquot of the washed cells was taken for PCR amplification with the linkage primers (Fusion R, Fusion F, 27f and HD9 - Table 2). The PCR cycle used was Program 1 (Table 3) and the PCR Master Mix components were as given in Table 3 (Stage 4, Figure 1). The amplification product was then centrifuged to pellet cells. The cells were washed a further 3 times with PBS before resuspension in 10 microlitres of 1 X PCR buffer. A second PCR round was then carried out using primers 1492r, an "internal" primer to 27f, and HD9 only and Program 2 (Table 3) which gave a longer annealing time for the formation of the linkage fragment. Following PCR amplification the PCR product was centrifuged to separate the cells. The supernatant was analysed for PCR amplification products. The cells were washed with PBS, lysed and then analysed for PCR amplification products. Presence of the linked product within the PCR amplification products was confirmed by electrophoresis in a 1% (w/v) ethidium bromide stained agarose gel.

Table 2

PCR amplification primers for the linkage of 16S rDNA with dioxygenase gene in

Arthrobacter sp. cells.

Table 3

PCR cycling conditions for the linkage of 16S rDNA with dioxygenase gene in

Arthrobacter sp. cells.

EXAMPLE 3

Bacterial cells may be separated from soil particles by dispersion in a Waring blender with 100 mM sodium phosphate buffer (pH 7.0) for 1 min. Coarse particles may then be allowed to settle for 1 min before the resultant suspension is serially diluted to remove the remaining soil particles. Bacterial cells may then be attached to paramagnetic beads by antibodies specific to bacterial cell wall proteins and the beads washed to remove any residual traces of soil particles, which could interfere with further processing. The beads may then be serially diluted in a microtitre plate format to give approximately one cell per well. Using a gentle cell lysis solution (0.5 mg ml^"1 for 30 min at room temperature) the bacterial wall can be permeabilised such that primers and reagents are able to enter the cell but nuclear material is not able to escape. A first round of PCR, using primers 1, 2, 3 and 4 (Table 4), can then be carried out to create amplicons of both the 16S rRNA and amoA genes (cycling conditions - 10 cycles of 30 s at 94°C, 60 s at 55°C, and 45 s at 72°C).

Following amplification, the linker fragment, 1 unit of lambda exonuclease and 1 unit of Klenow fragment (3 '-5') may be added (15 min at 37°C). The action of lambda exonuclease will promote the formation of 3' overhangs on both the linker fragment and the amplicons of the 16S rRNA and amoA genes. A hybrid molecule of the three DNA strands will then be formed from the overlapping binding sequences for primers 2 and 3 on the linker and amplicon strands where the gaps will be filled by the Klenow fragment. Following construction of the hybrid molecule it will no longer be necessary for the cell to remain intact and therefore a conventional PCR (cycling conditions - 60 s at 94°C followed by 30 cycles of 30 s at 94°C, 60 s at 55°C, 45 s at 72°C, followed by 5 min at 72°C) may be carried out using the primer set 1 (16S rRNA for ammonia oxidiser) and 4 (amoA for ammonia oxidiser). The amplified product can then be sequenced using internal sequencing primers.

Table 4

Oligonucleotide probe sequences suitable for amplification and linkage of phylogenetic and functional genes in environmental samples.

Name Sequence (5'-3') Target Reference

Primerl TGGGGRATAACGCA 16S rDNA of β-subgroup McCaig et al, 1994 β AMOf YCGAAAG ammonia oxidising bacteria

Primer 2 AGACTCCGATCCGG 16S rDNA of β-subgroup McCaig et al, 1994 βAMOr ACTACG ammonia oxidising bacteria

Primer 3 GGGGTTTCTACTGG AmoA gene of ammoma Rotthauwe et al, α oA-lF TGGT oxidising bacteria 1997

Primer 4 CCCCTCKGSAAAGC AmoA gene of ammonia Rotthauwe et al, amo A-2R CTTCTTC oxidising bacteria 1997

Claims

Claims:

1. A method for genetic analysis of a microbial sample by linking taxonomic to functional characteristics, the method comprising co-amplification of a taxonomic gene and a functional gene in a microbial sample and linking of the two amplified products.

2. A method of analysing a microbial sample comprising introducing intracellularly reagents for effecting co-amplification of a taxonomic gene and a functional gene if present in the cell, applying amplification conditions, applying conditions for linking any amplified products of the taxonomic gene with amplified products of the functional gene, and analysing for the linked product.

3. A method according to claim 1 or 2 carried out on intact cells.

4. A method according to any of claims 1 to 3 preceded by the steps of:

(a) extraction of cells from an environmental sample;

(b) immobilisation of intact extracted cells on a solid support; and

(c) semi-permeabilisation of the immobilised cells to enable entry of reagents for amplification.

5. A method according to claim 4 in which the isolated cells are immobilised on beads, the beads washed and placed in a well or matrix, under serial dilution to provide one cell per well/matrix.

6. A method according to any of claims 1 to 5 in which the microbial sample is a culture of one or more microbes or an enrichment culture.

7. A method according to any one of claims 1 to 6 in which the microbial sample is a natural microbial sample such as a soil or oil sample.

8. A method according to claim 7 in which the sample contains unculturable microbes.

9. A method according to any preceding claim in which the primers for the taxonomic gene are 16S rDNA primers.

10. A method according to any of claims 1 to 9 in which the primers for the functional gene are specific for a gene of interest.

11. A method according to any of claims 1 to 10 in which the functional gene primers are random primers that may be used to amplify previously unknown genes.

12. A method according to any of claims 1 to 11 in which the linking of the two amplified products is accomplished with a linker molecule

13. A method according to any of claims 1 to 12 comprising the steps of:

(a) providing a microbial sample with primers for a taxonomic gene and a functional gene;

(d) co-amplification of the hybrid product; and

(e) detection and analysis of the amplified hybrid product.

14. A method according to either of claims 11 or 13 in which linking is carried out by a linker molecule which has one end which is complementary to a recognition site in a taxonomic gene primer and another end which is complementary to a recognition site in a functional gene primer.

15. A method according to any of claims 11, 12, 13 or 14 in which linking is done in the presence of lambda exonuclease and Klenow fragment (3'-5').

16. A method according to any of claims 1 to 11 wherein the amplification product of the taxonomic gene has a "tail" complementary to a tail of an amplification product of the functional gene and the amplified products are linked by hybridisation of said tails.

17. A method as claimed in claim 16 wherein the linked product is amplified intra-cellularly.

18. A method as claimed in claim 17 wherein the linked product is amplified extra-cellularly.

19. A method according to claim 1 further comprising amplification of the hybrid product with one primer specific for the taxonomic gene fragment and one primer specific for the functional gene product.

20. A method according to claim 1 comprising:

(b) providing primers for a taxonomic gene fragment and a functional gene fragment, the primers carrying extension sequences x' and y\ which are capable of hybridising to sequences x and y on the solid support;

(d) linking the amplification products at x' and y' to provide a hybrid product comprising the taxonomic gene fragment, x', y' and the functional gene product; and

(e) amplifying the hybrid product.

21. A method according to claim 16 in which linkage of x' to y' is carried out using DNA ligase.