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PubMedDate not supplied

Species-specific polymerase chain reaction amplification of camel (Camelus) DNA extracts.

A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.

Animals↗
PubMed1991

PCR-based typing of DNA extracted from cigarette butts.

Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

DNA↗
PubMed2003

Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction.

Competitive approaches have shown promise for overcoming some of the difficulties in the use of PCR for assessment of specific bacterial species in soil. A competitive touchdown PCR (cTD-PCR) protocol specific for the rrsB gene of Escherichia coli was developed for tracking the organism in environments impacted by human wastes. Regression of product ratios from co-amplification of varying amounts of analyte and competitor DNA templates was linear. To test the robustness of the method, reactions were titrated with an extract of sterilized soil; no significant effect was detected. The cTD-PCR was used to assay recovery of E. coli DNA from soil. Stock DNA was spiked onto two sterilized soils during extraction, and the purified extracts were analyzed by cTD-PCR. Recovery of DNA spiked at a rate of 180 ng g(-1) was 34+/-7% (mean+/-S.D.) for an agricultural silt loam. DNA spiked at 1.8 pg g(-1) was recovered at a mean rate of 6.1+/-1.3%. DNA in these extracts was not directly quantifiable by image analysis. The cTD-PCR method provides a useful means of quantifying small amounts of E. coli DNA, and could be modified for other specific targets in a mixture of DNA from a variety of organisms.

Blotting, Southern↗
PubMed2001

Detection of bacteria in environmental samples by direct PCR without DNA extraction.

Cultured cells and environmental samples were used directly in PCRs without the isolation of DNA. Serial dilution was used to eliminate the inhibitory effect of materials in natural samples. Primers specific for pmoA, which encodes a subunit of the particulate methane monooxygenase, were used to detect and quantify methanotrophic bacteria by direct most probable number PCR. Phototrophic bacteria were detected in environmental samples by direct PCR with primers specific for pufM, and members of the bacterial domain were detected with primers for 16S rDNA. Direct PCR provides a rapid, simple, and sensitive methodfor detecting and quantifying bacteria in environmental samples. Detection of methanotrophic bacteria can be applied to monitoring bioremediation.

Bacteria↗
PubMed2001

Rapid detection of smear-negative Mycobacterium tuberculosis by PCR and sequencing for rifampin resistance with DNA extracted directly from slides.

Conventional methods for identification of Mycobacterium tuberculosis from culture can take 6 weeks. To facilitate the rapid detection of M. tuberculosis and to assess the risks of drug resistance, we developed a technique of eluting DNA directly from sputum slides and performing PCR for the detection of M. tuberculosis DNA, followed by sequencing the rpoB gene to detect rifampin resistance. This entire process requires only 48 h. Forty-seven sputum specimens submitted for microscopy for detection of acid-fast bacilli (AFB) and for mycobacterial culture and susceptibility testing were assessed after elution from the slides and extraction. M. tuberculosis-specific DNA was amplified in a nested PCR with previously described primers (primers rpo95-rpo293 and rpo105-rpo273), followed by analysis on a 4% agarose gel for a 168-bp product. Automated sequencing was performed, and the sequences were aligned against a database for detection of anomalies in the rpoB gene (codons 511 to 533) which indicate rifampin resistance. Of the 47 sputum specimens tested, 51% (24 of 47) were culture positive (time to positive culture, 2 to 6 weeks). Smears for AFB were positive for 58% (14 of 24) of the specimens and were negative for 42% (10 of 24) of the specimens. All 24 culture-positive sputum specimens (14 microscopy-positive and 10 microscopy-negative sputum specimens) were positive by PCR with eluates from the smears. Forty-nine percent (23 of 47) of the sputum specimens were negative for M. tuberculosis by smear, culture, and PCR. Of the isolates from the culture-positive samples, five were rifampin resistant by sequencing; all five were also rifampin resistant by in vitro susceptibility testing. Of these rifampin-resistant M. tuberculosis isolates, two were microscopy negative for AFB. Patients who are negative for AFB and culture positive for M. tuberculosis can now be identified within a day, allowing institution of therapy and reducing isolation time and medical costs.

Antitubercular Agents↗
PubMed1998

DNA extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction.

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol BD. In the first procedure, DNA is isolated from 1-20 microL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 microL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.

Cellulose↗
PubMed1979

Nucleotide sequence of the self-priming 3' terminus of the single-stranded DNA extracted from the parvovirus Kilham rat virus.

The parvovirus genome is a linear, single-stranded DNA molecule with double-stranded hairpin termini. The 3' terminus can serve in vitro as a self-primer for the synthesis of a double-stranded viral DNA intermediate. We have sequenced the nucleotides in the 3' terminus and propose a model for the secondary structure of the terminus and the in vitro origin of replication for the complementary viral DNA strand.

Base Sequence↗
PubMed1998

Rapid DNA extraction method for genetic screening.

A fast, simple, safe and inexpensive method for purification of genomic DNA from blood samples for subsequent PCR is described. All steps are carried out in a single 1.5 ml reaction tube. Neither proteinase K digestion nor precipitation steps are involved. The method is highly efficient. As little as 20 microl of whole blood gives enough DNA for reliable amplification by PCR. The blood cells are lysated in a buffer, the nuclei are pelleted and resuspended in water after a wash step. The resuspension is directly used for PCR.

DNA↗
PubMed2001

Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells.

The sensitivity of non-isotopic PCR-SSCP was compared to direct sequencing of PTEN exons. DNA from leukocytes derived from healthy donors, and from the glioblastoma cell line LN319 was extracted and mixed in different proportions from 0 to 100%. The LN319 cell line contains a point mutation at codon 15 exon 1 of the PTEN gene. The PCR-SSCP experiments demonstrated mutations in samples containing as little as 10% tumor DNA. In contrast, direct DNA sequencing experiments were less sensitive, requiring 30-70% of tumor DNA in the sample, depending on the DNA strand sequenced. In conclusion, PCR-SSCP, in our hands, is more sensitive than automated sequencing for detecting PTEN point mutations. We recommend to always sequence both strands, and take into account that samples containing less than 30% tumor cells should not only be sequenced, but also studied by PCR-SSCP in order to discriminate false negative results.

Brain Neoplasms↗
PubMed2005

Rapid and simple procedure for homogenizing leaf tissues suitable for mini-midi-scale DNA extraction in rice.

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi-scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3+/-1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 microg from 25-150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.

DNA, Plant↗