Inosine at Different Primer Positions to Study Structure and Diversity of Prokaryotic Populations

Culture-independent methods, employed to study the diversity and complexity of microbial communities that are based on amplification of rRNA genes with universal primers, include gradient gel electrophoresis (denaturing or temperature), single-strand-conformation polymorphism, restriction fragment length polymorphism, qPCR and highthroughput DNA sequencing. Substituting one or more base(s) within or at the 3’-termi of the universal primers by inosine can overcome some of their shortcomings improving amplification capacity. Universal primer sets do not usually amplify sequences with nucleotide mismatch to the templates, particularly in the last three bases, whereas inosine-modified primers anneal and amplify a variety of rRNA gene sequences. Inosine-containing primers are therefore might be useful to detect more species in diverse prokaryotic populations. The article summarizes the pros and cons of using inosine especially at the 3' termini of universal primers in nucleic acid amplification for the study of microbial diversity. Introduction Phylogenetic analysis and study of prokaryotic diversity based on 16S rRNA gene comparison (Woese and Fox, 1977) was enhanced by discovery of the PCR (Weisburg, et al., 1991). Culture-independent methods are advantageous to investigate the occurrence and distribution of bacteria in nature, providing direct information on community structure (Daly et al., 2000; Steven et al., 2007; Su et al., 2012). Most of these studies require extraction of total bacterial DNA from the sample and PCR amplification of one of the rRNA genes using universal primers designed according to their conserved sequences. This approach is straightforward, but biased at almost all steps: DNA extraction, primers selection and amplification (Bru et al., 2008; Fujiwara et al., 1995; Hansen et al., 1998; MartinLaurent et al., 2001; Polz and Cavanaugh, 1998; Suzuki and Giovanni, 1996; Wu et al., 2009), producing an incomplete and often distorted view (Forney et al., 2004) of the community structure. The choice of universal 16S rRNA gene primers used in studies to assess the diversity of prokaryotes is not trivial becouse (Baker et al., 2003; Forney et al., 2004): (a) complementarity to a large fraction of the sequences in databases such as the ribosomal database project [RDP; (Cole et al., 2009)] are not necessarily optimal; (b) sequences in the database may be incomplete or ambiguous; (c) no current data base faithfully represents the estimated total number of over 10 million bacterial species, with possible high sequence divergence (Curtis et al., 2002; Winsley et al., 2012). Efforts are therefore being made to improve universality of the primers. Substituting canonical bases by inosine Inosine is identical to guanine lacking the N2 amino group. It is found in the 5'-nucleotide of tRNA anticodons of mRNA, known as Watson–Crick wobble position, when different triplets encode the same amino acid (Crick, 1966), and loosely pairs to either cytosine, adenosine or uridine. Inosine's annealing intensities to the four nucleotides depends on the thermodynamic stabilities of the pairings (Martin et al., 1985; Watkins and SantaLucia, 2005). Nevertheless, it is successfully used in a variety of PCR primers and probes that require degeneracy such as at the wobble position, to permit annealing to different but closely related sequences (Candrian et al., 1991; Ohtsuka et al., 1985). The annealing temperature and the composition of reaction mixtures can be manipulated to achieve better universality of such inosine containing primers. The presence of inosine in an oligonucleotide seems to neither disturb nor destabilize DNA duplex formation (Ohtsuka et al., 1985). Replacing inosine (I) in degenerate primers often yields amplification results superior than of the mixedbase degenerate primers (Liu and Nichols, 1994). In internal positions of synthetic oligonucleotides, pairing of I to thymine (T) or guanine (G) reduces the hybrid yield 10-30 fold and to a lesser extent to adenine (A) over its pairing to cytosine (C) (Case-Green and Southern, 1994). The order of stabilities of paired-inosine is: I·C > I·A > I·T ≈ I·G > I·I (Martin et al., 1985); it is thus the favorite base to replace two (G/T, G/A, T/A), three or four base ambiguities. At the 3'-end, I pairs preferentially in the same order, albeit with reduced discrimination between the four bases, but at the 5'-end, it shows the same signal strength when paired against each of them (Case-Green and Southern, 1994). The hierarchy of hybridization yields of an octamer with two adjacent I's in the center is CC > CA > AA > AC > GC> GA > CG > TA > TC > CT = AG > AT > GT > TT (Case-Green and Southern, 1994). A neighboring pair has large influence on the stability of I·N base pair at the 5'or 3'-end of an oligonucleotide with the following tendency's hierarchy: G·C > C·G > A·T > T·A, reflecting a complex interplay between H-bonding, nearest-neighbor stacking and mismatch geometry (Watkins and SantaLucia, 2005). Curr. Issues Mol. Biol. (2015) 17: 53-56. horizonpress.com/cimb #53 Inosine at Different Primer Positions to Study Structure and Diversity of Prokaryotic Populations Inosine and Primers Universality Ben-Dov and Ariel Kushmaro This information may be useful in designing a primer/probe of optimal stability (Martin et al., 1985; Watkins and SantaLucia, 2005) to enhance primer universality while retaining specificity. While these parameters can be modulated in an attempt to increase universality, it is likely to be accompanied by a tradeoff in specificity so that nontarget sequences are also amplified (Forney et al., 2004). Utility of inosine in culture-independent methods Inosine at internal primer positions Two types of primers are used to enhance their universality for amplification of related sequences from different species having more than one nucleotide at a given location: degenerate primers or those containing I (Kilpatrick et al., 1996; Rossolini et al., 1994; Wu et al., 2009). Replacing a wobble-position base by I substantially reduces unspecific annealing of degenerate primer sets commonly used for amplification of protein-coding or of highly-conserved (e.g., of rRNA) genes. Introducing residues of I into the core of universal primers homologous to conserved regions in the 16S rRNA genes, for example, enabled amplification and detection of phylotypes that were not detected using the original, unmodified primers (Watanabe et al., 2001). Such primers were used in analyses of microbial diversity of oil palm rhizosphere (Acevedo et al., 2014), cattle manure and anode biofilms (Inoue et al., 2013). I-containing primers for different types of human papillomavirus yield higher amounts and more specific amplicons than the corresponding degenerate primers (Rossolini et al., 1994). Internally-placed I in primers for 16S rRNA were recently used in next-generation DNA sequencing to catalog bacterial reads within complex, polymicrobial specimens (Salipante et al., 2013). A large proportion of commonly used universal primers for 16S rRNA genes lack sequence homology to many of the “candidate” divisions, severely limiting bacterial variety assessments (Winsley et al., 2012) and thus display diverse coverage rates (Wang and Qian, 2009). So-called “conserved regions” in the 16S rDNA used as universal primers include many mismatches in the core (Watanabe et al., 2001; Thomas et al., 2011) and less in their 3'-end (Ben-Dov et al., 2011; Brands et al., 2010). Inosine at the 3'-end of primers Inclusion of degenerate bases or I at the 3 ́-end is usually considered undesirable because incompatible annealing of the ultimate or penultimate base(s) can suffice to initiate PCR at wrong sites (White, 1993). On the other hand, mismatched nucleotides at these positions may be detrimental to the amplification process (Kwok et al., 1990; Sarkar et al., 1990; Wu et al., 2009) because loose primer hybridization affects binding stability of the DNA polymerase to a primer-template terminus (Detera et al., 1981; Huang et al., 1992; Kamtekar et al., 2006). This is primarily due to a need for a perfect 3'-end base pair to allow enzymatic synthesis (Batzer et al., 1991). A point mutation at this position in the template will therefore have a much greater effect than mutations in neighboring bases. Inclusion of I at the 3'-end position in PCR primers of the Alu family, seminally performed by Batzer at al. (1991), resulted in a 150 bp amplicon from green monkey DNA, which was not amplified using identical primers without I. Using I at the 3'-termini of universal primers for 16S rDNA to study microbial diversity discovered 7 bacterial phyla whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (Ben-Dov et al., 2006). I-substituted universal primers for 16S rDNA at the 3'termini were used successfully in analyses of microbial diversity from different environmental niches such as industrial wastewater (Shapiro et al., 2009), coral black band diseases microbial mats (Barneah et al., 2007), desert soil (Angel et al., 2009), grapevine leaves (Bulgari et al., 2009), human feces (Patil et al., 2012; Zheng et al., 2009), oral plaque biofilms (Brands et al., 2010), hot spring ecosystems (Valverde et al., 2012) and gut flora of sand flies (Mukhopadhyay et al., 2012). This increased diversity confirms the usefulness of I-containing 3'-termini primerpairs to expand the observed diversity of microbial communities but is not guaranteed to amplify all species existing in the examined sample. An additional approach to obtain expanded microbial diversity uses substitutions of the two last bases at the 3′end of each of the primers by two inosines (Ben-Dov et al., 2011). This primer-pair expands somewhat the observed diversity of a bacterial community at the family/class level, but significantly shifts the composition of the resulting 16S rDNA libraries from that obtained by two other pri


Introduction
Phylogenetic analysis and study of prokaryotic diversity based on 16S rRNA gene comparison (Woese and Fox, 1977) was enhanced by discovery of the PCR (Weisburg, et al., 1991).Culture-independent methods are advantageous to investigate the occurrence and distribution of bacteria in nature, providing direct information on community structure (Daly et al., 2000;Steven et al., 2007;Su et al., 2012).Most of these studies require extraction of total bacterial DNA from the sample and PCR amplification of one of the rRNA genes using universal primers designed according to their conserved sequences.This approach is straightforward, but biased at almost all steps: DNA extraction, primers selection and amplification (Bru et al., 2008;Fujiwara et al., 1995;Hansen et al., 1998;Martin-Laurent et al., 2001;Polz and Cavanaugh, 1998;Suzuki and Giovanni, 1996;Wu et al., 2009), producing an incomplete and often distorted view (Forney et al., 2004) of the community structure.
The choice of universal 16S rRNA gene primers used in studies to assess the diversity of prokaryotes is not trivial becouse (Baker et al., 2003;Forney et al., 2004): (a) complementarity to a large fraction of the sequences in databases such as the ribosomal database project [RDP; (Cole et al., 2009)] are not necessarily optimal; (b) sequences in the database may be incomplete or ambiguous; (c) no current data base faithfully represents the estimated total number of over 10 million bacterial species, with possible high sequence divergence (Curtis et al., 2002;Winsley et al., 2012).Efforts are therefore being made to improve universality of the primers.

Substituting canonical bases by inosine
Inosine is identical to guanine lacking the N2 amino group.It is found in the 5'-nucleotide of tRNA anticodons of mRNA, known as Watson-Crick wobble position, when different triplets encode the same amino acid (Crick, 1966), and loosely pairs to either cytosine, adenosine or uridine.Inosine's annealing intensities to the four nucleotides depends on the thermodynamic stabilities of the pairings (Martin et al., 1985;Watkins and SantaLucia, 2005).Nevertheless, it is successfully used in a variety of PCR primers and probes that require degeneracy such as at the wobble position, to permit annealing to different but closely related sequences (Candrian et al., 1991;Ohtsuka et al., 1985).The annealing temperature and the composition of reaction mixtures can be manipulated to achieve better universality of such inosine containing primers.The presence of inosine in an oligonucleotide seems to neither disturb nor destabilize DNA duplex formation (Ohtsuka et al., 1985).Replacing inosine (I) in degenerate primers often yields amplification results superior than of the mixedbase degenerate primers (Liu and Nichols, 1994).In internal positions of synthetic oligonucleotides, pairing of I to thymine (T) or guanine (G) reduces the hybrid yield 10-30 fold and to a lesser extent to adenine (A) over its pairing to cytosine (C) (Case-Green and Southern, 1994).The order of stabilities of paired-inosine is: (Martin et al., 1985); it is thus the favorite base to replace two (G/T, G/A, T/A), three or four base ambiguities.At the 3'-end, I pairs preferentially in the same order, albeit with reduced discrimination between the four bases, but at the 5'-end, it shows the same signal strength when paired against each of them (Case- Green and Southern, 1994).
The hierarchy of hybridization yields of an octamer with two adjacent I's in the center is CC > CA > AA > AC > GC> GA > CG > TA > TC > CT = AG > AT > GT > TT (Case-Green and Southern, 1994).A neighboring pair has large influence on the stability of I•N base pair at the 5'-or 3'-end of an oligonucleotide with the following tendency's hierarchy: G•C > C•G > A•T > T•A, reflecting a complex interplay between H-bonding, nearest-neighbor stacking and mismatch geometry (Watkins and SantaLucia, 2005 Ben-Dov and Ariel Kushmaro This information may be useful in designing a primer/probe of optimal stability (Martin et al., 1985;Watkins and SantaLucia, 2005) to enhance primer universality while retaining specificity.While these parameters can be modulated in an attempt to increase universality, it is likely to be accompanied by a tradeoff in specificity so that nontarget sequences are also amplified (Forney et al., 2004).

Utility of inosine in culture-independent methods
Inosine at internal primer positions Two types of primers are used to enhance their universality for amplification of related sequences from different species having more than one nucleotide at a given location: degenerate primers or those containing I (Kilpatrick et al., 1996;Rossolini et al., 1994;Wu et al., 2009).Replacing a wobble-position base by I substantially reduces unspecific annealing of degenerate primer sets commonly used for amplification of protein-coding or of highly-conserved (e.g., of rRNA) genes.
Introducing residues of I into the core of universal primers homologous to conserved regions in the 16S rRNA genes, for example, enabled amplification and detection of phylotypes that were not detected using the original, unmodified primers (Watanabe et al., 2001).Such primers were used in analyses of microbial diversity of oil palm rhizosphere (Acevedo et al., 2014), cattle manure and anode biofilms (Inoue et al., 2013).I-containing primers for different types of human papillomavirus yield higher amounts and more specific amplicons than the corresponding degenerate primers (Rossolini et al., 1994).Internally-placed I in primers for 16S rRNA were recently used in next-generation DNA sequencing to catalog bacterial reads within complex, polymicrobial specimens (Salipante et al., 2013).
A large proportion of commonly used universal primers for 16S rRNA genes lack sequence homology to many of the "candidate" divisions, severely limiting bacterial variety assessments (Winsley et al., 2012) and thus display diverse coverage rates (Wang and Qian, 2009).So-called "conserved regions" in the 16S rDNA used as universal primers include many mismatches in the core (Watanabe et al., 2001;Thomas et al., 2011) and less in their 3 '-end (Ben-Dov et al., 2011;Brands et al., 2010).

Inosine at the 3'-end of primers
Inclusion of degenerate bases or I at the 3´-end is usually considered undesirable because incompatible annealing of the ultimate or penultimate base(s) can suffice to initiate PCR at wrong sites (White, 1993).On the other hand, mismatched nucleotides at these positions may be detrimental to the amplification process (Kwok et al., 1990;Sarkar et al., 1990;Wu et al., 2009) because loose primer hybridization affects binding stability of the DNA polymerase to a primer-template terminus (Detera et al., 1981;Huang et al., 1992;Kamtekar et al., 2006).This is primarily due to a need for a perfect 3'-end base pair to allow enzymatic synthesis (Batzer et al., 1991).A point mutation at this position in the template will therefore have a much greater effect than mutations in neighboring bases.
Inclusion of I at the 3'-end position in PCR primers of the Alu family, seminally performed by Batzer at al. (1991), resulted in a 150 bp amplicon from green monkey DNA, which was not amplified using identical primers without I.Using I at the 3'-termini of universal primers for 16S rDNA to study microbial diversity discovered 7 bacterial phyla whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (Ben-Dov et al., 2006).I-substituted universal primers for 16S rDNA at the 3'termini were used successfully in analyses of microbial diversity from different environmental niches such as industrial wastewater (Shapiro et al., 2009), coral black band diseases microbial mats (Barneah et al., 2007), desert soil (Angel et al., 2009), grapevine leaves (Bulgari et al., 2009), human feces (Patil et al., 2012;Zheng et al., 2009), oral plaque biofilms (Brands et al., 2010), hot spring ecosystems (Valverde et al., 2012) and gut flora of sand flies (Mukhopadhyay et al., 2012).This increased diversity confirms the usefulness of I-containing 3'-termini primerpairs to expand the observed diversity of microbial communities but is not guaranteed to amplify all species existing in the examined sample.
An additional approach to obtain expanded microbial diversity uses substitutions of the two last bases at the 3′end of each of the primers by two inosines (Ben-Dov et al., 2011).This primer-pair expands somewhat the observed diversity of a bacterial community at the family/class level, but significantly shifts the composition of the resulting 16S rDNA libraries from that obtained by two other primer-pairs, one with a single I substitution at the 3'-end and the other with no substitution.The most obvious shift in composition came with the detection of a higher abundance of sequences related to the class Clostridia.When two I's substituted AG in the two last 3′-end bases of the forward primer, the resulting hybridization (I•T-I•C) apparently destabilized primer-template-polymerase complex, and since the base adjacent to the 3'-end in the 16S rDNA of Clostridia's template contains a high frequency of G's (98.8%) the formation of first pairing (C•G) by Taq polymerase stabilized the initial amplification efficiency and thus biased it toward this class (Ben-Dov et al., 2011).
The base adjacent to the 3′-end of universal PCR primers targeting 16S rDNA is variable and thus impacts the initial phase of PCR amplification: A or T decrease PCR efficiency whereas G or C stabilize primer-templatee n z y m e -n u c l e o t i d e c o m p l e x a n d t h u s i n c r e a s e amplification rates (Ben-Dov et al., 2012).I-modified primers preferentially (but not exclusively) amplify sequences containing G or C next to the 3′-end of the primer over sequences containing A or T (Brands et al., 2010).Such next-base effects can distort data used to draw conclusions about structures of microbial communities in a PCR-based approach, due to bias in estimating the relative ratios between phylotypes having different template-contiguous bases to the 3′ end primers (Ben-Dov et al., 2012).

Concluding remarks
High-throughput or 'next generation' DNA sequencing platforms dramatically increase sequencing depth of phylogenetic analyses of explored microbial communities, but limitation in universality of commonly used primers can still provide only a narrow view of these communities.PCR amplification of DNA is a key preliminary step in many applications of high-throughput sequencing technologies, Ben-Dov and Ariel Kushmaro yet design and taxonomic analysis of novel universal or near-universal primers remains a challenging task (Walters et al., 2011).Universal primers containing inosine can reveal more varied templates and thus expand the observed diversity of microbial communities, but does not necessarily amplify all heterologous sequences of different species existing in explored niches.The importance of adopting multiple approaches to design universal primers for PCR analyses to assess microbial biota in complex environments is emphasized and substantiated.