Patent Application: US-44807806-A

Abstract:
the invention disclosed relates to a pcr primer pair for amplication of chaperonin - 60 targets having high g + c content and to a pcr primer “ cocktail ” to improve the representation of diverse sequences in chaperonin - 60 based pcr product libraries derived from complex templates . in previous cpn60 - based and 16s rdna - based studies of mammalian intestinal microbiota , it has been observed that some classes of organisms such as the actinobacteria , which are known through culture - based studies to be present in large numbers in these environments , are underrepresented or even absent from pcr product libraries . using library sequence data and reference cpn60 sequence data from cpndb , the chaperonin sequence database , we designed a pair of pcr primers which can be used alone for higher g + c content targets and , when used in combination with a previously developed degenerate , universal cpn60 primer pair , improve the representation of complex templates with high g + c content . we have validated these primers using a combination of traditional and quantitative real - time pcr on both artificially constructed complex templates and biological samples . the development and optimization of this primer cocktail represents a significant advance in our ability to generate cpn60 pcr product libraries which more closely represent the biodiversity in complex microbial communities .

Description:
as used herein , “ purified ” does not require absolute purity but is instead intended as a relative definition . for example , purification of starting material or natural material to at least one order of magnitude , preferably two or three orders of magnitude is expressly contemplated as falling within the definition of “ purified ”. as used herein , the term “ isolated ” requires that the material be removed from its original environment . described herein is a primer pair for amplification of cpn60 targets having high g + c content comprising : a first primer comprising a nucleotide sequence as set forth in seq id no . 1 ; and a second primer comprising a nucleotide sequence as set forth in seq id no . 2 . in other embodiments , the first primer may consist essentially of a nucleotide sequence as set forth in seq id no . 1 ; and the second primer may consist essentially of a nucleotide sequence as set forth in seq id no . 2 . in yet other embodiments , the first primer may consist of a nucleotide sequence as set forth in seq id no . 1 ; and the second primer may consist of a nucleotide sequence as set forth in seq id no . 2 . in some embodiments , the target has a g + c content of at least 32 %. in other embodiments , the target has a g + c content of about 32 % to about 71 %, from about 32 % to about 68 % or from about 32 % to about 63 %. in another aspect of the invention , the above - described primers are used in a method of amplifying partial cpn60 sequences from genomic dna comprising : b ) adding a primer pair for amplification of cpn60 targets having high g + c content comprising : a first primer comprising a nucleotide sequence as set forth in seq id no . 1 ; and a second primer comprising a nucleotide sequence as set forth in seq id no . 2 ; and examples of conditions suitable for nucleotide amplification are well - known to those of skill in the art and include enzymes , buffers , nucleotides , incubation times and temperatures and the like . exemplary pcr conditions are shown for example in the description of fig4 although other suitable conditions for nucleotide amplification will be readily apparent to one of skill in the art . in some embodiments , a second primer pair comprising adding a third primer comprising a nucleotide sequence as set forth in seq id no . 3 and a fourth primer comprising a nucleotide sequence as set forth in seq id no . 4 is added to the mixture prior to step ( c ). the third primer may consist essentially of a nucleotide sequence as set forth in seq id no . 3 and the fourth primer may consist essentially of a nucleotide sequence as set forth in seq id no . 4 . in other embodiments , the third primer may consist of a nucleotide sequence as set forth in seq id no . 3 and the fourth primer may consist of a nucleotide sequence as set forth in seq id no . 4 . as will be appreciated by one of skill in the art , as used herein , ‘ first primer ’, ‘ second primer ’, ‘ third primer ’ and ‘ fourth primer ’ are relative terms and serve to identify the primers but do not indicate a required order of addition . as discussed below , the first primer and the second primer may be added to the sample at a concentration at a ratio of from 1 : 1 to 10 : 1 compared to the concentration of the third primer and the fourth primer in the sample . specifically , the ratio may be from 1 : 1 to 10 : 1 or from 1 : 1 to 9 : 1 or from 1 : 1 to 8 : 1 or from 1 : 1 to 7 : 1 or from 1 : 1 to 6 : 1 or from 1 : 1 to 5 : 1 or from 1 : 1 to 4 : 1 or from 1 : 1 to 3 : 1 or from 1 : 1 to 2 : 1 . this mixture comprising two primer pairs can be used for amplification of partial cpn60 sequences from organisms of both high and low g + c content as discussed below . examples of suitable bacterial targets include those bacteria having a conserved cpn60 sequence which can be amplified under suitable conditions using primer pairs h279 / h280 and / or h1612 / h1613 . suitable bacterial targets will be well known to one of skill in the art and exemplary examples of such are provided herein for example in table 1 . examples of suitable samples include but are by no means limited to soil , sludge , waste water , bodily fluids , intestinal contents and the like . as discussed below , the primer pair described herein can be used alone or in combination with h279 / h280 for many purposes , for example , for following population dynamics of a complex bacterial community , determining effectiveness of antimicrobials , determining the effect of feeding regimes on gut flora and the like . typically , a primer pair includes a primer which is complementary to the negative (−) strand of the polymorphic locus , and the other is complementary to the positive (+) strand . the availability of a large reference data set of cpn60 sequences ( hill et al ., 2004 ) made it possible to study the primer annealing sites in organisms with high g + c content . a phylogenetic group of organisms can have a characteristic g + c content ; however , g + c content can also vary within a phylogenetic group . phylogenetically distant organisms can have identical g + c contents . we aligned 155 full - length cpn60 sequences ( 54 genera ) with g + c contents of at least 58 %, identified the primer annealing sites for h279 and h280 and calculated the frequency of occurrence of a , t , g and c at each position ( fig1 ). by comparing the high g + c template primer annealing sites with h279 and h280 and considering frequencies of at least 10 % to be significant , we designed the novel pcr primer pair h1612 and h1613 . the net result was the reduction from 9 to 5 inosine residues in the forward primer and 6 to 1 inosine in the reverse primer . the degeneracy of the forward primer increased from 2 to 16 and the reverse primer decrease in degeneracy from 64 to 16 ( table 1 ). inosine can base pair with each of the 4 naturally occurring bases . in the original primers , inosine residues were introduced at positions in the sequence where all 4 bases occurred . alternatively , introducing all 4 naturally occurring bases at these sites would have produced a degeneracy of 4 at each site since the primer pool synthesized would include 4 distinct sequences at a given site . if 2 sites were completely degenerate , the total degeneracy would be 16 fold ; if 3 sites , the total would be 64 fold etc . going in the reverse direction , if we changed an inosine residue to an a or a g , we would increase the degeneracy of the primer by 2 fold . the following data summarizes experimental evidence demonstrating that the oligonucleotide primers h11612 ( seq id no . 1 ) and h11613 ( seq id no . 2 ) (“ strong primers ”) can be used by themselves to amplify partial cpn60 sequences from dna templates ranging in g + c content from ( at least ) 32 % to 63 %. oligonucleotides h11612 and h11613 were designed based on the analysis of cpn60 sequences from source organisms with g + c content of at least 58 %. the primers anneal to templates in the same regions as h279 ( seq id no . 3 )/ h280 ( seq id no . 4 ) and amplify a region of the cpn60 gene corresponding to nucleotides 274 - 828 of the escherichia coli cpn60 gene . in the process of validating the primers for pcr use , we applied them to individual bacterial templates ranging in g + c content from 30 % to 63 %. as illustrated in fig2 , primers h279 / h280 amplified cpn60 sequences from the first 3 templates but failed to amplify from templates with g + c content of & gt ; 53 %. primers h1612 / h1613 amplified all templates with the exception of brachyspira hyodysenteriae ( g + c content of 30 %). primers h1612 / h1613 fail to amplify some low g + c templates and they may fail to amplify many or all templates with similarly low g + c contents . we also applied primers h11612 / h1613 to mixtures of dna templates with a range of g + c contents to determine if all of the component templates would be amplified and thus be detectable in the pcr product mixture produced . a mixture of 5 bacterial genomic dna samples was created according to the following proportions : 27 % clostridium perfringens , 9 % lactobacillus gasseri , 33 % enterococcus faecalis , 14 % aeromonas salmonicida and 17 % bifidobacterium animalis . pcr reactions were performed using either primers h279 / h280 or h1612 / h1613 . the amount of pcr product corresponding to each template in the reaction products after 40 cycles of pcr was determined using quantitative real - time pcr . as illustrated in fig3 , after amplification with the h279 / h280 , pcr products from the a . salmonicida and b . animalis templates ( 59 % and 62 % g + c respectively ) were not detected , indicating that these templates were not amplified with h279 / h280 . however , after amplification with the h1612 / h1613 primer combination , all templates could be detected in the pcr product pool . the results presented in fig2 and 3 demonstrate that the h1612 / h1613 primer pair can be used to amplify partial cpn60 sequences from genomic dna templates ranging in g + c content from 32 % to 63 %. we anticipate , based on sequence analysis and experiments to date , that they will work on even higher g + c contents . specifically , we have used these primers to amplify cpn60 sequences with a 68 % g + c content from an environmental organism present in muck soil that was amended with fish emulsion . in order to test the efficacy of novel primer pair h1612 / h1613 in combination with the h279 / h280 primer pair , we created a mixture of genomic dna from 5 species , ranging in cpn60 g + c content from 32 % to 62 % ( table 2 ). three independent mixtures of the 5 species were made for 4 replicates of the amplification experiment . the composition of each mixture was determined by quantitative pcr using the species - specific primers described in table 1 , using a standard curve generated from a dilution series of cloned cpn60 pcr products for each species ( dumonceaux et al 2005 ) and the proportion of the total population comprised by each of the species was calculated ( table 3 ). sample mixtures were amplified with various ratios of pcr primers h279 / h280 : h1612 / h1613 ( 10 : 0 , 7 : 1 , 3 : 1 , 1 : 1 , 1 : 3 , 1 : 7 , and 0 : 10 molar ratios ) at each of 4 annealing temperatures ( 42 ° c ., 46 . 5 ° c ., 50 . 4 ° c ., 56 ° c .) and the products were pooled . to create complex microbial community libraries , we routinely do the pcr amplifications over a range of temperatures since we have previously demonstrated that annealing temperature affects library composition ( hill et al ., 2002 ) and our goal is to maximize diversity in the library . pcr products derived from each of the 5 templates were quantified using the species - specific pcr primers shown in table 1 and expressed as proportions of the total pcr product mixture . fig4 shows the relative representations of each of the 5 templates pre - and post - amplification . the aeromonas salmonicida and bifidobacterium animalis templates which had each accounted for 10 - 20 % of the original template mixtures were undetectable by quantitative pcr in experiments where the h279 / h280 primer pair was used exclusively . as the relative amount of h1612 / h1613 primer increased to a 1 : 3 ratio , the representation of these g + c - rich templates improved . further skewing of the primer cocktail ratio beyond 1 : 3 ( to 1 : 7 and 0 : 10 ) did not improve the representation of these templates . after amplification with a 1 : 3 ratio of h279 / h280 : h1612 / h613 , the proportions of each template in the amplified pcr product population were within a log of their proportions in the original template . to demonstrate the effectiveness of the primer cocktail ( 1 : 3 ratio of h279 / h280 : h1612 / h1613 ) for amplification of sequences from a natural microbial community , we obtained total genomic dna from fecal samples from 6 pigs . using species - specific pcr primers we quantitated the amount of l . amylovorus and b . animalis in the samples ( fig5 a ). in agreement with culture - based studies of pig fecal microbial populations , we found that these targets were present at approximately 10 to 10 genomes per gram . after pcr amplification with a 10 : 0 ratio of h279 / h280 : h1612 / h1613 primers ( fig5 b ), we conducted quantitative real - time pcr with species - specific pcr primers to determine the relative amounts of l . amylovorus and b . animalis targets in the pcr product pool . while the l . amylovorus target was easily detectable in the primary pcr product pool , we only detected b . animalis amplicons in one of two independent amplifications from pig 243 and the small amount detected was at the extreme lower limit of the standard curve (˜ 10 3 copies ). in contrast , an examination of the pcr product pool produced using the 1 : 3 ratio of h279 / h280 : h1612 / h1613 primers showed that b . animalis pcr products were present in relative quantities to the l . amylovorus target similar to that observed in the starting material ( fig5 c ). a library created from the pcr product pool created with the 1 : 3 cocktail of h279 / h280 : h612 / h11613 ( fig5 c ) would be much more likely to contain b . animalis sequences than a library created from the pcr product pool created with the h279 / h280 primers alone ( fig5 b ). in fact , a cpn60 library recently created from pig intestinal contents using the primer cocktail presented here does in fact contain bifidobacterium sequences . an essential first step in the study of a microbial community is an inventory of its constituents . this inventory can subsequently be used in the design of molecular tools for the quantification and monitoring of population members . our observations indicate that application of the 1 : 3h279 / h280 : h1612 / h1613 primer cocktail will allow the construction of cpn60 pcr product libraries that more accurately represent the diversity of a microbial population than was possible with the h279 / h280 primers alone . these libraries will provide an excellent starting point for more intensive studies of microbial population dynamics . environments that we have examined include the microbiota of soils that suppress plant pathogens , the microbiota of the human vagina , the microbiota of activated sludge waste water in pulp and paper mills , the microbiota in intestinal tracts of animals fed control diets or diets that included antimicrobials . other suitable environments will be apparent to those of skill in the art . while the preferred embodiments of the invention have been described above , it will be recognized and understood that various modifications may be made therein , and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention . benno , y ., endo , k ., suzuki , k ., mitsuoka , t ., and namioka , s . 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