The degenerate primer design problem: theory and applications.

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly degenerate primers for a set of genomic sequences. We report on the success of the program in several applications, one of which is an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.     

The degenerate primer design problem

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly-degenerate primers for a set of genomic sequences. We report on the success of the program in an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.     

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy

Cytochrome P-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. An efficient PCR strategy for isolating cytochrome P-450 cDNA clones from plant cDNA libraries is described. A set of degenerate primers for PCR amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome P-450 proteins. Using this primer set and a non-specific primer, complementary to either the poly(A) tail of the cDNA clones or a phage vector sequence, we isolated 16 different cytochrome P-450 cDNA sequences from a cDNA library of Catharanthus roseus.     

Isolation of segments of homologous genes with only one conserved amino acid region via PCR

We present a method which allows the isolation of fragments from genes coding for homologous proteins via PCR when only one block of conserved amino acids is available. Sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences. The second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by DNA synthesis. The cassette is ligated to partially-digested chromosomal DNA. The second primer is biotinylated to allow elimination of PCR products carrying degenerated primers on both sides via streptavidin binding. Fragments obtained after amplification and enrichment are cloned and sequenced. The feasibility of this method was demonstrated in a model experiment, where degenerated primers were deduced from six conserved amino acids within the family of homologs to the Escherichia coli Vsr protein.     

从水稻Ac/Ds插入突变体扩增Ds侧翼序列的最适TAIL-PCR引物

温度非对称交互PCR(TAIL-PCR)技术已广泛应用于从多种生物体系克隆侧翼于已知序列的DNA片段的分子操作中,并极大地促进了反向遗传学研究。但是,可能由于不同物种间基因组大小和序列存在显差异,在采用该技术进行转座元件Ds水稻插入突变体鉴定过程中,常因TAIL-PCR反应的稳定性差而影响突变体筛选效率。有鉴于此,根据Ds核苷酸序列设计了分别对应或互补于Ds插入元件两端长度不同的12个特异引物组成32个组合,在大量预试验基础上与6个不同简并性(32～256)的随机简并引物分别组合进行TAIL-PCR反应,较系统地研究了引物特性对以水稻基因组DNA为模板的TAIL-PCR反应效率的影响。结果发现,第一反应采用长序列特异引物(36～40mer)可显提高扩增特异性,随机简并引物的简并度对反应的影响显。还选择出两个适于从水稻Ds插入突变体基因组高效扩增出Ds插入侧翼片段的最优特异引物组合和最适简并引物。应用本研究结果可显地提高TAIL-PCR技术筛选水稻插入突变体的效率。     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.     

EasyExonPrimer

EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. AVAILABILITY: EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. CONTACT: Xiaolin Wu (forestwu@mail.nih.gov).     

Reliable amplification of actin genes facilitates deep-level phylogeny

The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.     

Ketosynthase Domain Probes Identify Two Subclasses of Fungal Polyketide Synthase Genes

Analysis of fungal polyketide synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketosynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketosynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal polyketide synthase genes.     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.     

Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.     

Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches

The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

New molecular screening tools for analysis of free-living diazotrophs in soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Cross-species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

We have developed and optimized a consistent polymerase chain reaction (PCR)-based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time-consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3'-untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand-alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.