IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis
© Wilkins et al; licensee BioMed Central Ltd. 2006
Received: 15 September 2006
Accepted: 19 December 2006
Published: 19 December 2006
Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps.
Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: (1) an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; (2) detection of the Mopti and Savanna rDNA types; and (3) an assay to distinguish resistance to dieldrin (Rdl) alleles in Anopheles arabiensis.
Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands.
The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.
Single nucleotide polymorphisms (SNPs) have been utilized as markers for species differentiation  and are implicated in insecticide resistance in many anopheline mosquitoes . While various polymerase chain reaction (PCR) approaches to discriminate SNPs exist, specific primers for the wild-type and/or mutant alleles are often designed to perfectly match the sequence of one allele whereas the second varies only by matching the alternative SNP sequence at the terminal 3' end. Therefore, it is critical that the biochemical specificity of the two primers is sufficient to determine the success of PCR amplification from a particular template. This PCR-specificity depends on two characteristics: the ability of any mismatch to prevent extension of the primer by the polymerase – many polymerases can synthesize template over a single terminal mismatch  – and the difference between the annealing temperature (Tm) of the perfect and mismatched primer . For both of these reasons, a single 3' mismatch has limited discrimination. In this report, intentional mismatches were used to increase the discrimination of PCR based primarily on the polymerase synthesis characteristic.
The Anopheles gambiae complex is comprised of six genetically and behaviourally distinct species that are morphologically almost identical . Members of this complex have been identified as major vectors of human malaria parasites, and as many as four species may be sympatric. Several methods for identifying these species have been developed, such as gas chromatography of cuticular hydrocarbons , polytene chromosome arrangements (reviewed in ) and allozyme analyses . However, none of these has obtained as widespread use as PCR amplification of the ribosomal DNA (rDNA). This has become the standard method for species discrimination since rDNA polymorphism detected by Southern hybridization  spurred development of a PCR assay . While the Scott et al. PCR method is the most popular, non-specific bands and high rates of PCR amplification failure were observed in our hands.
In addition to the cryptic species of the An. gambiae complex described above, karyotype analysis of the female ovarian chromosomes has revealed further subdivision into chromosomal forms called Forrest, Mopti (M) and Savanna (S) . The observed frequencies of the inversions that define these types are correlated with different ecological zones suggesting that they may have an adaptive character. Because karyotype analysis is tedious and can only be performed on semi-gravid females, several alternative PCR-based methods have been developed. PCR amplification of the rDNA intergenic spacer IGS [10–12] and internal transcribed spacers ITS1 and ITS2  of An. gambiae s.s. have provided a molecular surrogate for karyotypes of Mopti and Savanna types in some but not all areas . The novel method reported here was compared with the diagnostic PCR-RFLP method of Fanello  which amplifies a portion of the IGS followed by digestion of the products with Hha I to produce diagnostic fragments of either rDNA type.
Resistance to dieldrin (Rdl) in An. gambiae s. l. is due to a single nucleotide mutation within the M2 transmembrane subunit of γ-aminobutyric acid (GABA) receptor . In Anopheles arabiensis, resistance is conferred by an alanine to serine substitution within the Rdl locus. Resistance to dieldrin per se (a cyclodiene) is of little immediate concern since it has been banned from use in public health and agriculture. However, recently it has been shown that cross-resistance to the phenylpyrazole fipronil in Anopheles stephensi and An. gambiae was also due to the presence of an Rdl resistance allele [15, 16].
In this report, intentional mismatch primers – IMPs- a method previously utilized by Papp et al.  – were used to introduce mismatches at the third nucleotide from the 3' end. This simple modification of intentionally adding a mismatch to the SNP-detection primers counter-intuitively increases the specificity of the primers, which is demonstrated here for three anopheline SNP detection analyses.
An. gambiae s.s
Primer sequences. Lower case nucleotide indicates the intentional mismatch, nucleotides in bold are located at site of SNP (where applicable), F and R indicate forward and reverse orientation.
An. gambiae species ID
(5' to 3')Fragment (size bp)
An. quadriannulatus (637)
An. melas and merus (529)
An. gambiae (464)
An. arabiensis (388)
Mopti/Savanna rDNA typing
Rdl mutation assay
positive control (256)
dieldrin resistant (158)
dieldrin susceptible (121)
An. gambiae species identification PCR cycling consisted of melting at 95°C for 5 min followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, followed by one cycle of 72°C for 5 min. Mopti and Savanna rDNA were analysed using PCR-amplified rDNA and Hha I digests by the method of Fanello . Hha I digests were performed overnight at 37°C. The locations of novel primers were selected at SNP sites using published sequence data [Genbank: AF470116 and AF470112]. PCR thermal cycling for the IMP primers consisted of melting at 95°C for 5 min followed by 30 cycles of 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds, followed by one cycle of 72°C for 5 min.
An. arabiensis Rdl-specific primers were designed to be used with the previously reported An. gambiae internal positive control primers RDLF and RDLR . Thermal cycling consisted of melting at 95°C for 5 min followed by 30 amplification cycles of 95°C for 45 seconds, 53°C for 45 seconds, and 72°C for 45 seconds, followed by one cycle of 72°C for 10 min. Heterozygous F1 individuals were produced for analysis by crossing KGB (homozygous susceptible) females to SENN (homozygous resistant) males en masse.
Results and discussion
In this study, only laboratory colonies of mosquitoes were tested for species ID, and validation on a larger scale as has been performed previously for Anopheles quadrimaculatus s.l.  will be necessary to confirm their utility. In order to ensure that the IMP primer sets would function correctly in strains other than those we analysed and for which sequence data was available, reported sequences were examined [1, 12, 21–23] and no polymorphisms were found that would interfere with the success of the protocol.
The novel method, upon which these assays is based, is that all SNP-specific primers were designed to contain an intentional mismatch for both target alleles at the third nucleotide from the 3' end and with the terminal base falling on the site of the SNP . These primers were tested over a wide range of annealing temperatures using a gradient cycler and were found to amplify their targets consistently and clearly over annealing temperatures of 50–65°C. This bodes favorably for reproducibility of the results obtained here on different machines. Successful amplification over a wide range of temperatures also suggests that the primer design is successful not due to a thermodynamic change leading to a lower Tm but rather due to the inability of a standard Taq polymerase to extend over a series with alternating mismatches at the 3' end.
Successful amplification using IMPs was achieved with all mosquito stages tested including larvae, pupae, and adult mosquitoes without extensive DNA purification. Therefore, these assays are useful for studies in the field where DNA preparation is not possible or time-prohibitive. The ease of design, technical simplicity, high specificity, and sensitivity recommend these as improved specific assays and the general method as a means to produce assays for both multicopy and single-copy SNPs for laboratory and field analyses. There is no apparent reason that similar primer designs would not be useful for analysis of human and Plasmodium SNPs and any application in which rapid sensitive SNP discrimination is necessary.
We appreciate the American Type Culture Collection and the NIAID-funded contract N01-AI-85355 for their financial and moral support for the operation of the MR4 and their cooperation in developing and implementing the subcontract to the CDC Foundation that makes this research possible. Special thanks to all the donors of mosquito stocks without whose collegial generosity these experiments would not have been possible: Maureen Coetzee (KGB and SKUQUA), Vincent Corbel (KISUMU1), Mustafa Dukeen (SENN), Greg Lanzaro (MOPTI), and Rajendra Maharaj (OPHANSI). Thanks to Lynn Huynh for the careful editing and discussion of this manuscript.
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