Direct Polymerase Chain Reaction Amplification Research Articles

Evaluation of Silica Resins for Direct and Efficient Extraction of DNA from Complex Biological Matrices in a Miniaturized Format

For DNA purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (μSPE) device containing only nanograms of silica resin is shown to be effective for the adsorption and desorption of DNA in the picogram–nanogram mass range. Fluorescence spectroscopy as well as capillary electrophoresis with laser-induced fluorescence detection is employed for the analysis of DNA recovered from solid-phase resins, while the polymerase chain reaction (PCR) is used to evaluate the amplifiable nature of the eluted DNA. We demonstrate that DNA can be directly recovered from white blood cells with an efficiency of roughly 70%, while greater than 80% of the protein is removed with a 500-nl bed volume μSPE process that takes less than 10 min. With a capacity in the range of 10–30 ng/mg of silica resin, we show that the DNA extracted from white blood cells, cultured cancer cells, and even whole blood on the low microliter scale is suitable for direct PCR amplification. The miniaturized format as well as rapid time frame for DNA extraction is compatible with the fast electrophoresis on microfabricated chips.

Comparison of Effects of Acidic Electrolyzed Water and NaOCl on Tilletia indica Teliospore Germination.

Definitive identification of free teliospores of Tilletia indica, causal agent of Karnal bunt of wheat, requires polymerase chain reaction (PCR)-based diagnostic tests. Since direct PCR amplification from teliospores has not been reliable, teliospores first must be germinated in order to obtain adequate DNA. We have routinely surface-sterilized teliospores for 2 min with 0.4% (vol/vol) sodium hypochlorite (NaOCl) to stimulate germination and produce axenic cultures. However, we observed that some spores were killed even with a 2-min NaOCl treatment, the shortest feasible duration. Decreasing the NaOCl concentration in our study from 0.4% to 0.3 and 0.2%, respectively, increased teliospore germination, but treatment times longer than 2 min still progressively reduced the germination percentages. In testing alternative methods, we found "acidic electrolyzed water" (AEW), generated by electrolysis of a weak solution of sodium chloride, also surface-sterilized and increased the rate of T. indica teliospore germination. In a representative experiment comparing the two methods, NaOCl (0.4%) for 2 min and AEW for 30 min increased germination from 19% (control) to 41 and 54%, respectively, by 7 days after treatment. Because teliospores can be treated with AEW for up to 2 h with little, if any, loss of viability, compared with 1 to 2 min for NaOCl, treatment with AEW has certain advantages over NaOCl for surface sterilizing and increasing germination of teliospores of suspect T. indica.

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains

From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification. The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity. By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification. A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains.

From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification. The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity. By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification. A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

Expression pattern of mammalian cochlea outer hair cell (OHC) mRNA: screening of a rat OHC cDNA library.

The aim of this study was to characterize the mRNA content of mammalian cochlear outer hair cells (OHCs) and to search for specific genes possibly involved in their unique properties. Indeed, OHCs, which feature high-frequency electromotility, are responsible for the exquisite sensitivity and frequency selectivity of the cochlea. Damage to these cells, which occurs in various conditions, causes a reduction in the cochlear sensitivity by about 50 dB and the alteration of frequency discrimination. Total RNA was extracted from about 2000 mechanically dissociated OHCs, and a polymerase chain reaction (PCR) amplified cDNA library was constructed. The presence of the alpha-9 acetylcholine receptor subunit, preferentially expressed in OHCs, was found by direct PCR amplification of the library. A systematic sequencing of 218 clones showed 78% known genes, 11% EST-related sequences, and 11% unknown genes. The known-gene group was characterized by two main features: a large proportion (55%) of mitochondrial transcripts and an abundance in calcium-binding proteins, such as calmodulin and calbindin, for which expression has already been demonstrated in OHCs. Another protein, the oncomodulin recently shown to be OHC specific, was also found, and its mRNA expression was confirmed by in situ hybridization. Among the 24 unknown genes, 7 were expressed in a restricted pattern, including one expressed in cochlea and spleen and, to a lesser extent, in lungs.

The Exon Structure of the Human MAGP-2 Gene: SIMILARITY WITH THE MAGP-1 GENE IS CONFINED TO TWO EXONS ENCODING A CYSTEINE-RICH REGION

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.

Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community.

A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils of different texture. DNA and rRNA yields from these soils were 15–30 and 0.25–1.0 μg g −1 soil, respectively. Following different purification steps, the rRNA as well as genomic DNA extracts obtained were sufficiently pure for either reverse transcription and polymerase chain reaction (PCR) amplification, or direct PCR amplification. Using a set of universal bacterial primers based on conserved regions of the 16S rRNA sequence, both approaches yielded mixed target molecules for subsequent denaturing gradient gel electrophoresis fingerprinting of soil microbial diversity. The amplified rRNA-based bacterial diversity assessment was compared with diversity assessments based on amplified DNA in one selected soil. Results showed similarities as well as differences between the profiles generated on the basis of rRNA and those based on genomic DNA, which suggested that the bacterial communities defined on the basis of their genomic DNA contained variable amounts of rRNA.

Species-specific polymerase chain reaction for the differentiation of larvae from Dictyocaulus viviparus and Dictyocaulus eckerti

Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10 000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.

Comparison of direct PCR and PCR amplification after DNA extraction for the detection of viable enterotoxigenic Escherichia coli in laboratory microcosms

Studies of laboratory microcosms have shown that enterotoxigenic Escherichia coli (ETEC) lose their culturability but remain viable when exposed to starvation conditions. A polymerase chain reaction (PCR) procedure was used to detect viable cells incubated in artificial seawater microcosms, either directly after boiling the water or after extracting the DNA. After 4 days of incubation, culturable bacterial counts decreased to < 1 CFU ml −1 but viable bacteria were detected for up to 6 days in all cases. After 15 days, only direct PCR remained positive and after 21 days, all results were negative. The non-culturable state of ETEC may pose health problems, and this study demonstrated the efficiency of direct PCR to detect total viable starved Escherichia coli strains.

A simple method for detecting human polyomavirus DNA in urine by the polymerase chain reaction.

In order to develop a simple and sensitive method for detecting human polyomavirus DNA in the urine of patients by the polymerase chain reaction (PCR), it was found that the viral DNA could be released from urine by proteinase K and then amplified by PCR directly, without additional treatment such as ultracentrifugation or DNA extraction. Direct PCR amplification of viral DNA from urine was volume limited and 5 microliters of urine appeared to be the optimum amount for direct PCR amplification. When the urine volume was greater than 10 microliters, the results of PCR were inconsistent. However, the urine volume could be increased after dialysis to remove possible inhibitor(s) which may interfere with PCR. Direct PCR amplification of patient urine is convenient and eliminates several steps which can cause loss of DNA from the sample.

Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis ((Delta)F508)

To evaluate direct polymerase chain reaction amplification of mutation on single embryo cells for the routine preimplantation diagnosis of cystic fibrosis. Direct polymerase chain reaction amplification of mutation was performed to identify the cystic fibrosis delta F508 mutation in human blood DNA, single lymphocytes, embryos, and embryo cells obtained by biopsy. Preimplantation diagnosis was performed for a couple who were heterozygous carriers of the delta F508 mutation. Laboratory for preimplantation diagnosis in a reproductive medicine unit. Correct diagnosis of homozygous normal, heterozygous, and homozygous abnormal DNA of the cystic fibrosis delta F508 mutation. 45 blood samples (18 homozygous normal, 17 heterozygous, and 10 homozygous abnormal) and 204 single lymphocytes from known sources showed 100% amplification and were diagnosed correctly. 17 human embryos and 52 normal nucleated embryo cells obtained by single cell embryo biopsy also showed 100% amplification. After a miscarriage of the initial pregnancy (diagnosed at preimplantation to be homozygous normal) in the heterozygous carrier couple, fetal tissue was confirmed to be homozygous normal. Direct polymerase chain reaction amplification of mutation is a simple, fast, reliable test for the common cystic fibrosis mutation (delta F508) in blood DNA and single cells and should be applicable to routine programmes of general screening, maternal blood examination, and preimplantation diagnosis.

Polymerase chain reaction and non-radioactive gene probe based identification of mosquito larvicidal strains of Bacillus sphaericus and monitoring of B. sphaericus 1593M, released in the environment

In attempts towards an accurate monitoring in the environment, the status of the deliberate release of Bacillus sphaericus, a powerful mosquito larvicidal agent, as well as to evolve a rapid method of screening for potent isolates of B. sphaericus, we have developed a polymerase chain reaction (PCR) method of analysis. Using specific primers spanning the 5′ and 3′ ends of the coding sequences of these two mosquito larvicidal genes, a 1.3 and a 1.1 kb product from gene A and a 2.6 kb product from gene B have been amplified by PCR. The primers and the products amplified from them in PCR are highly specific for B. sphaericus. We used digoxigenin based non-radioactive chemiluminescence method for the detection of PCR products and the sensitivity of the method was high enough to detect the presence of 1 to 5 cells of B. sphaericus. A simple and inexpensive sample processing procedure has also been developed for direct PCR amplification of B. sphaericus DNA from field samples collected from areas where it had been applied. Several highly toxic to non-toxic strains of B. sphaericus were screened with these primers by PCR for the presence of either or both of these toxin genes. The results indicate that there is a good correlation between the presence of both genes with higher toxicity of the strains.

Direct sequence determination of the influenza B HA-1 gene after PCR amplification of clinical specimens from an infected volunteer

When clinical isolates of influenza A and B viruses are propagated in embryonated hens' eggs or tissue culture cells, different selective pressures in vitro result in specific amino acid substitutions in the haemagglutinin (HA) gene. A proportion of such viruses which lose a potential glycosylation site near the receptor binding region of the HA at amino acid positions 196–198 appear to have reduced virulence. Direct polymerase chain reaction (PCR) amplifications of cDNA and subsequent nucleotide sequence analysis of part of the HA-1 gene of the original infecting influenza B strain and the nasal wash material from an infected volunteer were performed. The nucleotide sequences of the viral HA-1 from the nasopharynx of the infected volunteer were the same as that of the original infecting strain. Antigenic analysis of both the original infecting virus and the viruses isolated from sequential samples collected from the volunteer, all of which were cultivated on Madin-Darby Canine Kidney (MDCK) cells and in embryonated hens' eggs, revealed variation in the HA of viruses only after egg adaptation. In particular, we describe the use of direct nucleotide sequencing techniques without the use of cloning strategies in order to determine the sequence of the HA-1 gene after direct PCR amplification of clinical nasal wash material.

Finger prick blood testing in Leber hereditary optic neuropathy.

Individuals from 33 unrelated Australian families with optic atrophy were screened for 10 different single base alterations in mitochondrial DNA (mtDNA) associated with Leber hereditary optic neuropathy (LHON) using direct polymerase chain reaction amplification of blood spots collected on Guthrie cards. This method using blood spots allows easily accessible screening for LHON mtDNA mutations with minimal biohazard risk and reduced expense in the storage and transport of specimens.

An efficient site-directed mutagenesis using polymerase chain reaction.

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.