Hemi Nested Pcr

[Gundenia Ransbottom]

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CDV is a highly contagious viral pathogen causing a lethal systemic disease in dogs and other carnivores. Several lineages or genotypes of CDV exist that are variously distributed throughout several continents. Legal or uncontrolled trading of animals may modify the epidemiology of CDV, introducing novel strains in CDV-nave areas or accounting for the resurgence of CDV in areas where vaccine prophylaxis was effective and successful to control the disease. A hemi-nested PCR system was developed to genotype strains of the major CDV lineages, America-1, Europe, Asia-1, Asia-2 and Arctic. The assay was tested using a collection of 27 laboratory and vaccine strains and of 36 field CDV strains. Distinct lineages could be differentiated by specific primers targeted to the H gene. The method could be useful for molecular epidemiological studies of CDV, providing a tool for large-scale studies, and for the diagnosis of vaccine-related disease.

A simplified hemi-nested reverse transcriptase polymerase chain reaction (hnRT-PCR) has been developed to determine specifically the European Bat Lyssavirus 1 (EBLV-1) nucleoprotein gene. The specificity of this method was determined by using the seven genotypes of lyssavirus by RT-PCR, Southern blot and sequence analysis. Compared to the rabies diagnostic methods, the hnRT-PCR showed a higher sensitivity for the detection of small amounts of EBLV-1 virus. In view of these results, we suggest this new hnRT-PCR should be performed for the epidemiological survey of bat colonies, also providing rapid detection and genotyping of EBLV-1 until now encountered in all naturally infected bats in France.

Hexaploid wheat (Triticum aestivum L.) possesses a large genome that contains 1.6 1010 bp of DNA. Isolation of a large number of gene sequences from complex gene families with a high level of gene sequence identity from genomic DNA is therefore difficult and time-consuming. Bacterial artificial chromosome (BAC) libraries can be useful for such work. Here we report on an efficient approach for rapid isolation and sequencing of the low molecular weight glutenin subunit gene family from the 'Glenlea' wheat BAC library via primer-template mismatch PCR using universal primers, primer walking using hemi-nested touchdown (TD) PCR, and followed by direct sequencing of PCR products.

For the primer-template mismatch PCR, the universal primers were designed based on conserved gene coding regions of consensus sequences. The effects of the universal primer-template mismatches on the efficiency of standard PCR amplification were investigated after assembly of sequences from different primers amplifying the same BAC clones. Single or multiple mismatches were observed at 5' terminal, internal and the penultimate position, respectively. These mismatches included the transition mispairs G:T, T:G, A:C and the transversion mispairs A:A, A:G, G:G, G:A. Two or more primer-template mismatches reduced PCR product yield approximately from 2-fold to 10-fold compared to PCR product yield without the primer-template mismatch. For the hemi-nested TD PCR, primers were designed based on the known sequences obtained and/or published. The hemi-nested TD PCR increased both specificity and yield by high and low annealing temperatures in two consecutive amplifications. Comparison of two methods for purifying PCR products prior to sequencing showed that purification using MultiScreen384-PCR filter plates had an advantage over ethanol purification because greater numbers of sequencing reactions could be performed from comparable volumes of PCR reactions.

This approach was fast, easy and cost-effective for isolation and sequencing of genes from complex gene families. It may be suitable for (i) isolation of other complex gene families and/or gene homologues from BAC libraries, (ii) for characterization of multi-copy repetitive elements pending availability of BAC libraries, and (iii) for filling in gaps in shotgun BAC sequencing.

Wheat gluten is composed of two major groups of proteins, monomeric gliadins and polymeric glutenins. Wheat glutenins consist of high molecular weight glutenin subunits (HMW-GSs) and LMW-GSs. Together, they are the major determinants of wheat bread-making quality. HMW glutenins have been extensively studied because of their importance in dough rheology and their relative ease of analysis at the protein and DNA levels. HMW glutenins represent a small family with a maximum of five of the six subunits expressed, while LMW glutenin genes are part of a much more complex gene family [13].

'Glenlea' is a Canadian Western Extra Strong (CWES) spring wheat variety. The LMW-i and LMW-s type genes in 'Glenlea' have been recently cloned using genomic DNA and developing seed cDNA libraries [15, 16]. To date, more than 90 cDNA and genomic DNA clones of LMW-GS genes have been published [17]. Most studies reported only a single or very few genomic DNA clones of LMW-GS genes that were isolated from a given wheat variety due to their complex compositions [15, 16]. To further our understanding of the LMW-GS gene family, a BAC library was constructed from 'Glenlea' [18]. Here we report an efficient method for rapid isolation of sequences of the complex LMW-GS gene family from the 'Glenlea' BAC library using universal primers for primer-template mismatch PCR, primer walking using hemi-nested touchdown PCR and direct sequencing of PCR products.

A total of 383 positive BAC clones were identified by screening the 24 high-density filters with the LMW-GS composite probe. Positive signals may include BAC clones containing gliadin sequences, because LMW-GS and gliadin sequences share approximately 60% similarities [13].

In order to confirm the identity of positive BAC clones containing LMW-GS genes, LMW-GS specific primers were designed. After PCR screening, 72 out of the 383 BAC clones produced one amplicon with at least one of the three primer pairs. Thirty-one BAC clones were identified by single primer pair, while 41 BAC clones were identified by using two or three primer pairs (Table 1). Fig. 1 shows eight representative BAC clones identified using the primer pair LMWF3/LMWR4.

In order to remove the remaining salts, unincorporated dNTPs and primers from the PCR reactions, we compared two PCR purification methods, i.e. MultiScreen384-PCR filter plate and ethanol precipitation, for their ability to produce large amounts of high quality template for sequencing reactions. The PCR product from a single PCR reaction purified using the ethanol precipitation was only sufficient for a single sequencing reaction, whereas the combined PCR product from two PCR reactions purified using the MultiScreen384-PCR filter plate was sufficient for five (PCR with primer-template mismatches, see below) to 16 (PCR without primer-template mismatch) sequencing reactions. Hence, the Multiscreen384-PCR filter plate provided better recovery of PCR products and was particularly efficient for weak amplifications with primer-template mismatches. Both PCR purification methods produced 100% successful sequencing reactions. The length of high quality sequences obtained using ethanol precipitation and MultiScreen384-PCR filter plate was 530 and 548 bases, respectively.

Given the large number of PCR products to be sequenced, we employed a direct sequencing approach using the same primers used for the PCR reactions. Direct sequencing of PCR amplicons is efficient and cost-effective. Therefore, direct sequencing is advantageous over the cloning approach which is time-consuming and costly.

The PCR products of the 72 BAC clones amplified using the three primer sets were directly sequenced. Single sequence length with Phred score greater than 20 could reach up to 630 bases with an average of 535 bases. A single LMW-GS sequence was isolated in 23 BAC clones using one of the three individual primers, whereas 29 BAC clones had two or three single sequences isolated by different primers. This indicated that the 29 BAC clones might contain a single or two LMW-GS gene(s), when different sequences were assembled into one or two contigs. The remaining 20 BAC clones had a LMW-GS pseudogene sequence or two LMW-GS sequences isolated by a single primer pair, which resulted in overlapping sequences. For these BAC clones with the overlapping sequences using one pair of primers, specific primers need to be designed to amplify individual LMW-GS genes.

Twenty-three high quality sequences of partial LMW-GS genes were isolated using the primer set LMWF11/LMWR11. Their length ranges from 556 to 711 bases with an average of 628 bases. A phylogenetic tree of the 23 sequences was constructed using MEGA3.1. The 23 sequences were clustered into six groups, which correspond to six different N-terminal sequences of LMW-m and LMW-s type genes (Fig. 2). Sequence alignment showed that the sequences from different BAC clones in the same groups were identical in the aligned regions, suggesting that these BAC clones may contain identical LMW-GS genes.

Phylogenetic tree of partial LMW-GS gene sequences using primer pair LMWF11/LMWR11 constructed using MEGA3.1. Bootstrap values are indicated; bootstrap percentages are based on 1000 iterations. The scale bar indicates the level of sequence divergence. DNA sequences with the same N-terminal amino acid sequence were clustered in the same group.

After we obtained partial sequences of the coding regions of LMW-GS genes from individual BAC clones using the universal primers, we first tried to perform primer walking using BAC DNA as template and specific primers close to the end the known sequence obtained. This method worked well for BAC clones containing single LMW-GS gene in many cases, but not for BAC clones containing two or more LMW-GS genes (data not shown). Then we tried to use TAIL PCR for the BAC clones containing two or more LMW-GS genes by designing three specific primers based on the known sequences obtained and one degenerated primer as described in Liu and Huang [9]. In many cases, either more than two PCR products were amplified or no amplicons were generated (data not shown). Thirdly, we designed primers based on 5' and 3' terminal sequences of the known LMW-GS gene sequence [GenBank: X13306] [19] combined with the above mentioned specific primers in order to generate PCR fragments from these BAC clones by primer-template mismatches. This method did not work for all BAC clones even though we optimized PCR conditions by decreasing the concentration of Mg+2 or/and using different annealing temperatures. After numerous attempts, we finally developed a novel PCR method, hemi-nested TD PCR combined with primer-template mismatches, for primer walking. As shown in Fig. 3A, standard PCR of seven BAC clones using the forward primer LMWF38d and the external reverse primer LMWR19b with annealing temperature at 50C generated multiple unspecific amplicons. Standard PCR using the same primers with annealing temperature at 60C did not produce amplification at all (data not shown). Fig. 3B shows the first round TD PCR amplification using a program in which the annealing temperature is progressively lowered from 65C to 50C by 0.5C every cycle, followed by 15 additional cycles at 50C. Faint single PCR product was observed for five BAC clones and two PCR amplicons were generated for BAC clone TaE0212J01. After the second round hemi-nested TD PCR using the same forward primer LMWF38d and the internal reverse primer LMWR19c with the same TD program, a single strong PCR product was amplified for the all seven BAC clones (Fig. 3C). When the amplicons of Figure 3A were amplified using the same primers and PCR conditions, the identical PCR products were observed but they were weaker than the amplicons in Figure 3C (data not shown). The hemi-nested TD PCR worked well for all the BAC clones irrespective of the presence of one or two LMW-GS genes. So far, two different LMW-GS gene sequences have been obtained from four BAC clones, respectively.

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