Early studies on the diversity of bacterial DNA from forest soil indicated a large discrepancy between culture-based and culture-independent diversity Torsvik et al. Thus, bacterial communities are now characterized by a variety of culture-independent approaches, mostly consisting of information derived from 16S rRNA gene sequences.
Using 16S rRNA gene clone libraries to identify individual bacteria in mixed populations has been a popular tool Beja et al. The increasing availability of high-throughput sequencing, particularly pyrosequencing, is driving migration to these more comprehensive approaches and revealing even higher bacterial diversity Dowd et al. Because of the expense and time-consuming nature of these inclusive techniques, the need remains for less intensive methods of interrogating the microbial biodiversity present in specific samples.
Alternative techniques for characterizing microbial communities include terminal-restriction fragment length polymorphism, automated rRNA intergenic spacer analysis, denaturing gradient gel electrophoresis DGGE , and temperature gradient gel electrophoresis Fromin et al.
DGGE, as used in molecular microbial ecology, is based on a series of discoveries and modifications since DNA duplex fragments of similar size migrate through an acrylamide matrix with constant mobility, but dissociation of the two strands leads to a considerable decrease in mobility through the gel.
DNA fragments of similar size but varying sequence migrate through an increasing gradient of formamide and urea with constant mobility until the fragment with the lowest melting point dissociates. Fragments of similar size but with base-pair substitutions affect the melting point sufficiently to effect separation. Larger DNA fragments would transition to partially melted form, while the higher-melting-point domains would remain helical.
By attaching a GC clamp at one end, the melting point of the terminal domain is sufficiently higher than the rest, allowing for detection of single-base substitutions Myers et al.
The slight differences in stacking interactions between adjacent bases cause melting at slightly different denaturant concentrations. Initial GC clamps were bp in length, and later workers developed shorter clamps, down to 40 bp Sheffield et al. DGGE was first applied to the study of bacterial diversity in the early s Muyzer et al. This approach allowed the study of complex microbial populations without the requirement for laborious processes such as culturing or clonal sequencing, both of which have been shown to have a number of limitations Hugenholtz et al.
During the use of DGGE for several projects, we began to suspect variation between repeat sets of equivalent GC-clamp primers. All were obtained from the same reputable supplier at different dates, and paired with primer R Teske et al. The sequence in italics represents the GC clamp. The sequence in bold represents the 16S rRNA-directed primer. The background was subtracted using a rolling disk set at 20, and band density at positions was converted to intensity per R f value between 0 and 1.
After normalizing for total intensity across lanes, data were input into the past software package and analyzed using multivariate principal component analysis Hammer et al.
A total of 10 clones from each primer set reaction were chosen at random for sequencing. Vector sequence and 16S rRNA gene sequences downstream of the respective primer were removed manually. The melting temperature T m was calculated with biomatch Promega , using the base-stacking algorithm.
Empirical observations of differences in DGGE profiles generated using separate GC-clamp primer batches lead us to suspect variation in performance of distinct batches. To compare the effect of a longer template-directed sequence, primer G1 had the same GC-clamp sequence but an rather than a base 16S rRNA gene sequence Muyzer et al. In order to exclude any variations apart from the primer sequence, a strict protocol was followed.
A single master mix without forward primer was prepared and split into five aliquots before addition of the primers. The amount of template DNA used was standardized, and all PCR reactions were run in the same thermocycler and at the same time to ensure equal temperature conditions.
Profiles obtained using the various primer sets appeared similar to each other. Principal component analysis of band intensities across R f values separated the bacterial communities into two groups, U and C, by the first component Fig. Importantly, profiles based on repeat synthesis of the same primer sequence N1—N3 were not identical, irrespective of the soil sample used Fig.
These results were found to be repeatable across three separate experiments data not shown. The band intensity at various R f values was analyzed by Principal Component Analysis b. Variations among DGGE profiles from different batches of GC-clamp primers lead us to investigate the primer sequence of amplicons. PCR product from each of the reactions was cloned and 8—10 clones were randomly selected for sequencing. Alignment of the primer region revealed evidence of near-integrity of the 16S rRNA gene portion of the primer Table 2.
Truncations of the GC clamp were the most common error found throughout all the primers, with nine out of 10 such errors for primer G1. The results indicated that batches of GC-clamp-bearing primers are associated with different degrees of sequence variation within the amplicon pool. It is not clear whether this is due to variation among copies of the primers within a batch or whether these errors are introduced during the replication process.
The key indicates any sequencing errors that occurred. In order to determine whether variation in length and base composition of the GC clamp would affect banding patterns, we adopted an in silico approach. The primer corresponding sequences Table 2 were merged to the V3—5 region of the B. Assuming 0. This would invariably lead to multiple bands per 16S rRNA gene sequence, and an overestimation of the diversity.
More importantly, the same sequence would yield different banding patterns for different primer batches. The effect of GC-clamp sequence and length variation on band position was then studied experimentally. The V3—5 region of three separate bacterial species of bacteria was amplified using the five sets of primers, and the products were resolved by DGGE.
Each lane contained more than one band Fig. Importantly, the profiles based on primer sets varied among each other Fig. One 16S rRNA gene sequence can, therefore, yield multiple bands. The number and distance between the bands appears to be influenced by the specific batch of primers. Three of the five primers N1—N3 used had an identical sequence design, but displayed deviation both in DGGE patterns and in sequence integrity.
This may be due to several adjacent guanosine residues in primer F1. Whether these deviations from the intended sequence occur during synthesis of the oligonucleotide or during the PCR process is unclear from the current results. DNA synthesizers reportedly experience difficulty adding multiple adjacent guanosine residues Sheffield et al. These structures could interfere during both oligonucleotide synthesis and PCR.
Products of primers N1—N3 and F1 lead to a lower degree of GC-clamp variation, and contain only one di-guanosine. Yet, these primers also yielded multiple bands in pure-culture DGGE of all three species, indicating a range of T m within the amplicon pool. In lieu of multiple guanosines, the GC clamps contained multiple cytosine residues, which would generate multiple guanosines in the reverse strand.
These multiple guanosines could form temporary quartets and four-stranded tetraplexes, leading to the observed aberrations and truncations in subsequent cycles. DGGE is a technique in which the variability of sequence is used to show the presence of certain types of microorganisms. Thus, any change in the primer sequence attached to the amplified region has the potential to affect the banding pattern of the DGGE. The B.
The deviation for an incorrectly assembled GC-clamp primer extended to a GC content of This large degree of difference would easily translate into multiple bands on a DGGE gel and result in multiple bands for each sequence in the sample. In the original publication describing a bp GC clamp, suggestions on the design of GC-clamp primers were made Sheffield et al. Despite the actual sequence not being crucial, inverted repeats and strings of consecutive G nucleotides should be avoided Sheffield et al.
Strings of G nucleotides would be problematic in the synthesis process Sheffield et al. Notice that it does not matter where in the last 5 bases the G or C base is in order for them to be referred to as a GC clamp. Binding between G and C bases is formed by three hydrogen bonds, compared to only two between adenine A and thymine T base pairs. Therefore, G and C base pairs are considered to have stronger binding than A and T base pairs.
Since G and C base pairs have superior binding, placing of these bases at the end of the primer will encourage complete primer binding. Doing so can actually have adverse effects by increasing the primer melting temperature Tm and reducing primer specificity. In sum, a GC clamp is often recommended during PCR primer design in order to encourage complete primer binding to the complementary template.
However, too many G or C bases especially at the end of primers can have negative effects. These PCR reactions were performed successfully, with differing levels of primer-dimer formation and overall efficiency. If something is going to fail, it is not usually the PCR. This is not mandatory; it's one of the characters of the primer which might improve your PCR reaction.
At the same time, avoid a sequential combination of GC in the last 6bp which might lead to self-dimers. Example: 5' GCGC-3' has higher chance of dimer formation than 5' GCCG-3' ; in such cases you can still use the latter primer sequence. Unless you are PCRing something you know to be challenging, just use primer3 and don't worry about it.
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