通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

DNA聚合酶与引物/模板的相互作用对PCR效率的影响

PCR是体外酶促合成特异DNA片段的一种方法,引物的优劣直接关系到PCR的特异性与成功与否。传统的PCR引物设计软件基本上忽略了DNA聚合酶与引物/模板的亲和性对PCR效率的影响。为揭示DNA聚合酶与引物/模板的相互作用是否对PCR的效率有影响,通过构建Taq DNA 聚合酶与不同序列引物/模板DNA相互作用的三维结构模型,采用MM/GBSA方法计算复合物的结合自由能,以结合自由能为参数,为人血清白蛋白基因(Human Serum Albumin gene,HSA gene)和结核杆菌pyrF基因(Mycobacterium tuberculosis pyrF gene)设计了PCR引物。PCR实验结果表明,引物的PCR效率与结合自由能相关：引物与聚合酶的结合自由能越低,PCR实验的效率相对越高。这说明DNA聚合酶与引物/模板的相互作用对PCR效率有重要影响。因此,引物/模板DNA与聚合酶的结合自由能可以作为PCR引物设计的新参数。     

The influence of quality of primers on the formation of primer dimers in PCR

Abstract

Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3′-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3′-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

Papillomaviruses and herpesviruses as selected risk factors in the etiopathogenesis of cervix neck cancer. II. Use of PCR for isolating DNA of human papillomavirus and Herpes simplex

The aims of the study were to compare polymerase chain reaction PCR with nucleic acid hybridisation HC in the routine diagnosis of HPV infections. Smears collected for PCR were digested for 24 hours using proteinase K. After DNA extraction 174 samples were tested by PCR with human bglobin primers PG04-GH20. The PCR products were separated in 2% agarose gel electrophoresis stained with ethidium bromide. In 80.6% of the samples 256 base pair DNA fragments were observed in the gel in UV light. These samples were tested by PCR with HPV primers MY09-MY11. In 40% of the samples the presence of HPV DNA was confirmed. Next we carried out PCR using a mixture of two pairs of primers bglobin PG04-GH20 and HPV MY09-MY11. DNA for this study was extracted from 24 samples in which the presence of human DNA was not confirmed in the first PCR test and from 7 untested samples. In 21 cases HPV DNA was found to be present in gel electrophoresis. The presence of HPV DNA was confirmed in 44.75% of the samples.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Troubleshooting in gene splicing by overlap extension

A step-wise method for cloning intron-containing genes from genomic DNA is described. The two exons of the human proinsulin gene were separately amplified in two steps using, in the first step, completely homologous primers. This reduces unwanted interactions between mismatched primers and a complex DNA template such as genomic DNA. The fragments were amplified in a second step polymerase chain reaction (PCR) using mismatched primers that incorporated additional bases complementary to the other exon, and these products were spliced together in a third step PCR.     

A new method of pebrine inspection of silkworm egg using multiprimer PCR

Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5'-TTGCTCACGG TAGATAACAA GAGAG-3' and 5'-TTGCTCACGG ACCAGGTAGG GAATG-3') were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.     

随机引物在分子生物学研究中的应用

随机引物指非特异序列的寡聚核苷酸作为DNA合成过程中的引物, 是相对于特异引物的概念.90年代它与PCR技术结合衍生了几项新技术:采用不同长度随机引物进行DNA指纹分析而衍生出的RAPD、AP-PCR及DAF方法; 进行mRNA多态分析的“差异显示”; 以及rPCR, T-PCR等技术. 以RAPD为例介绍了随机引物PCR的技术特点及其在分子生物学研究中的应用.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.     

Detection of pathogenic leptospires in animals by PCR based on lipL21 and lipL32 genes

Efficacy of primers capable of amplifying conserved outer membrane protein (OMP) genes i.e., lipL21 and lipL32 of Leptospira strains was tested for rapid and early diagnosis of the leptospirosis using a polymerase chain reaction (PCR). These OMP genes were found to be conserved in various leptospiral serovars viz., Canicola, Pomona, Icterohaemorrhagiae, Pyrogenes, Sejroe, Grippotyphosa, Ballum and Tarassovi as PCR products of 561 bp and 756 bp were obtained by PCR employing lipL21 and lipL32 based primers, respectively, in all these serovars. Absence of such amplicons in DNA extracted from Pasteurella, Campylobacter and Brucella confirmed the specificity of the primers. Serum and tissue samples collected from cattle, buffaloes and experimentally infected guinea pigs and calves were subjected to PCR using above primers as well as conventionally used primers G1/G2. All the sera and tissue samples, whether field samples or collected from experimentally infected animals, found positive for G1/G2 specific PCR were also positive for lipL21 and lipL32 specific PCR. The present study indicated that lipL21 and lipL32 based primers could be used for PCR based diagnosis of leptospirosis. Since G1/G2 primers are known not to amplify the DNA of Grippotyphosa, the use of primers employed in the present study could have an additional advantage in detection of cases of the disease.