Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)

Aims:  To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification–PCR amplification).
Methods and Results:  MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (Φ 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml⁻¹) of bacteria within 8 h including DNA isolation.
Conclusion:  MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato.
Significance and Impact of study:  The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes.     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-microl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 +/- 0.7) x 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.     

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The F?rster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.     

A sex determination protocol for the Iberian desman (<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>) based on a three primer amplification of <Emphasis Type="Italic">DBX</Emphasis> and <Emphasis Type="Italic">DBY</Emphasis> fragments with non-invasive samples

We have sequenced partial fragments of DBX and DBY genes of the endangered Iberian desman (Galemys pyrenaicus). The sequences were used to design a sex determination protocol for non-invasive samples based on a PCR reaction, using only three primers. This protocol allows the simultaneous amplification of two fragments, one corresponding to the DBX gene and the other to the DBY gene, both differing in size. To increase sensitivity on the detection of positive amplifications and on the determination of fragment size we use a fluorescently labelled primer. The protocol has been tested in DNA samples from hair and stool, revealing major difficulties in sexing faecal samples, but unambiguous sexing of hair samples.     

Array-based mutation detection of BRCA1 using direct probe/target hybridization

We describe here an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCA1 using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on an aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild-type sample, a two-step method was used to generate labeled multiplex polymerase chain reaction (PCR) amplification products of genomic regions containing the mutation sites. Amino allyl-dUTP, an amine-modified nucleotide, was incorporated during multiplex PCR amplifications and a monofunctional form of cyanine 3 dye was subsequently attached to the reactive amine group of the PCR products. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites. This work demonstrates that oligonucleotides chip-based analysis is a good candidate for efficient clinical testing for BRCA1 mutations when combined with the indirect strategy to prepare labeled target samples.     

Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.

A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.     

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.     

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.     

Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions

Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutations. PAP is a method for nucleic acid amplification in which 3' blocked oligonucleotides (P*) are activated by pyrophosphorolysis when annealed to the target template and subsequently extended. A key advantage to this technology is that PAP reactions produce little or no primer-dimer or false priming. As a result of this enhanced specificity, MD-PAP is easy to optimize. Herein, we utilize MD-PAP to determine gene dosage of each exon of the human factor IX gene by comparison with one endogenous internal control from the ATM gene. Estimated dosage is proportional to the actual template copy number over a minimum dynamic range from 1 to 16 copies. A blinded analysis detected 100% of 43 heterozygous deletions of exons in the human factor IX gene.     

Improved AFLP protocol using dual-suppression PCR and its application to species with large genomes

To improve the amplified fragment length polymorphism assay, dual-suppression PCR was introduced into the preamplification step of the assay. The dual-suppression PCR blocked completely the amplification of fragments with the same sequence (Bsp1407I-Bsp1407I or NlaIII-NlaIII) at both ends and amplified selectively fragments with different adaptor sequences (Bsp1407I-NlaIII) at each end. Two protocols, referred to as A and B, were established for species with medium- and large-sized genomes, respectively. Both protocols incorporated the dual-suppression PCR. Protocol A resulted in high-quality electrophoretic profiles for black cottonwood and rice, which have medium-sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.     

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.     

Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR.

The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube.     

Analysis of mutational spectra by denaturing capillary electrophoresis

The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test. Received: 18 August 1997 / Accepted: 12 January 1998