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Results for “genomic DNA contamination”

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At least 19 recordsLinked to original sources
PubMed1997

Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA.

Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity. Epithelial cells (1-10) could be detected in 1 mL of blood. Among the numerous CK18 genes homologous to the transcribed gene, at least two different processed pseudogenes exist that are highly homologous to each other and to the exons of the transcribed CK18 gene.

Biomarkers, Tumor↗
PubMed1992

Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.

Artifacts↗
PubMed2001

Quantification of Escherichia coli genomic DNA contamination in recombinant protein preparations by polymerase chain reaction and affinity-based collection.

This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg x mL(-1) of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg x mL(-1) and1 ng x mL(-1) genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.

Base Sequence↗
PubMed2000

Poly(A) cDNA-specific (PACS) RT-PCR: a quantitative method for the measurement of any poly(A)-containing mRNA not affected by contaminating genomic DNA.

We present a simple and efficient RT-PCR method for the detection and quantitation of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA and does not rely on exhaustive DNase digestion protocols. The technique described here requires the use of an antisense primer designed to contain 6-8 bp cDNA-specific sequence and an additional 17 Ts located on the 5' end to take advantage of the poly(A) tail. A second cDNA-specific sense primer can be used that does not need to be separated by intronic DNA sequence.

Animals↗
PubMed1999

Analysis of IGF2 gene imprinting in breast and colorectal cancer by allele specific-PCR.

The insulin-like growth factor II (IGF2) gene is imprinted with the paternal allele expressed and the maternal one silent. Loss of imprinting (LOI) of IGF2 has been suggested to play a role in the development of tumours, but the reported incidence of IGF2 LOI in tumours shows considerable variation, which may stem from different methodologies employed. In particular, partial digestion of reverse transcriptase-polymerase chain reaction (RT-PCR) products by restriction enzymes can lead to inaccurate measurements. To overcome the problem of partial enzymatic digestion, a novel method termed allele specific-polymerase chain reaction (AS-PCR) has recently been reported, which provides a significant advance over enzymatic digestion. A second problem with measurements of biallelic IGF2 transcription is that the co-amplification of contaminating genomic DNA during the RT-PCR step can lead to an overestimation of the frequency of biallelic IGF2 expression. To investigate the extent of this problem, total RNA from breast and colorectal cancer was analysed using two methods. The first method involved a first-round PCR using cDNA generated with primers spanning exons 8 and 9 (exon connection), followed by a second round of AS-PCR using primers from within exon 9. The second method used only AS-PCR with primers from within exon 9. The result was that the exon-connection approach was more accurate, thereby highlighting a significant problem in imprinting analyses where genomic DNA contamination cannot be completely ruled out.

Alleles↗
PubMedDate not supplied

P450 aromatase messenger ribonucleic acid expression in male rat germ cells: detection by reverse transcription-polymerase chain reaction amplification.

We have previously demonstrated that cytochrome P450 aromatase (P450arom) protein, an estrogen-synthesizing enzyme, is present and active in germ cells of the adult mouse testis. To establish that P450arom mRNA is expressed in germ cells of other species, we examined expression of P450arom in adult rat germ cells by employing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from Staput separated germ cells and reverse transcribed. The resulting cDNA was amplified by nested PCR reactions using oligonucleotide primers selected from a highly conserved region of the P450arom gene. RT-PCR analysis yielded cDNA products of 334 bp in length that corresponded to the predicted size expected from the final nested amplification. The identity of the germ cell P450arom PCR products was confirmed by restriction enzyme analysis and direct nucleotide sequencing. Rat genomic DNA was subjected to PCR to verify that P450arom DNA products were not obtained from genomic DNA contamination. Rat genomic DNA yielded a nested PCR product for P450arom of approximately 2000 bp, suggesting that, as is the case with the human P450arom gene, the rat P450arom gene contains an intron in the amplified region. In addition, a semiquantitative technique was utilized to eliminate the possibility that the P450arom RT-PCR products were derived from Leydig cell contamination of Sta-put-separated germ cell preparations. RT-PCR for P450arom and 3-beta-hydroxysteroid dehydrogenase (3 beta-HSD), a Leydig cell-specific steroidogenic enzyme, was carried out on Sta-put-separated germ cells and interstitial cell preparations containing Leydig cells. P450arom and 3 beta-HSD RT-PCR reactions were stopped at three cycle intervals to detect and compare the earliest appearance of RT-PCR reaction products in various cell types. Results indicated that P450arom mRNA is detected in round spermatids before it is detected in interstitial cells, whereas 3 beta-HSD was detected only in interstitial cells, suggesting that the P450arom mRNA detected in germ cells is not due to interstitial cell contamination of germ cell preparations. Therefore, our results indicate that P450arom mRNA is expressed in adult rat germ cells and that testicular germ cells are a potential source of estrogen in the male reproductive tract.

Amino Acid Sequence↗
PubMed2001

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.

Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.

Base Sequence↗
PubMed2003

A novel algorithm for computational identification of contaminated EST libraries.

A key goal of the Human Genome Project was to understand the complete set of human proteins, the proteome. Since the genome sequence by itself is not sufficient for predicting new genes and alternative splicing events that lead to new proteins, expressed sequence tags (ESTs) are used as the primary tool for these purposes. The high prevalence of artifacts in dbEST, however, often leads to invalid predictions. Here we describe a novel method for recognizing genomic DNA contamination and other artifacts that cannot be identified using current EST cleaning techniques. Our method uses the alignment of the entire set of ESTs to the human genome to identify highly contaminated EST libraries. We discovered 53 highly contaminated libraries and a subset of 24 766 ESTs from these libraries that probably represent contamination with genomic DNA, pre-mRNA, and ESTs that span non-canonical introns. Although this is only a small fraction of the entire EST dataset, each contaminating sequence could create a spurious transcript prediction. Indeed, in the clustering and assembly tool that we used, these sequences would have caused incorrect inference of 9575 new splice variants and 6370 new genes. Conclusions based on EST analysis, including prediction of alternative splicing, should be re-evaluated in light of these results. Our method, along with the identified set of contaminated sequences, will be essential for applications that depend on large EST datasets.

Algorithms↗
PubMed2006

A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer.

The aim of the present study was to decrease the incidence of false positives and to better characterize marginally cytokeratin-19 (CK-19) mRNA positive peripheral blood samples from patients with early stage breast cancer. A new set of highly specific primers for CK-19, which avoids amplification of contaminating genomic DNA, was designed and evaluated to improve the specificity and sensitivity of the previously described methodology. The primers were specifically designed to avoid amplification of contaminating genomic DNA and CK-19 pseudogenes. The breast cancer cell line MCF-7 was used as positive control for the development and analytical evaluation of the assay, while peripheral blood samples from 62 healthy female individuals and 160 patients with early breast cancer were used for the evaluation of the sensitivity and specificity of the new primer pair. The novel designed primer pair was highly sensitive, as it detects up to 1 MCF-7 cell, and specific as none of the healthy individuals had detectable CK-19 mRNA positive cells in their peripheral blood. CK-19 mRNA positive cells were detected in 33 out of 160 (20.6%) patients with early breast cancer. Results obtained by the proposed optimized real-time RT-PCR protocol correlated well with those obtained in the same samples by our previously reported quantitative real-time RT-PCR [concordance in 198/222 (89.2%), p = 0.0022, McNemar test]. The improved method eliminates the incidence of false positives and is highly sensitive and specific. The method could be used in a clinical setting in the near future for continuous monitoring and quantification of circulating epithelial cells in the peripheral blood of patients with operable breast cancer, provided that a quite larger number of clinical samples with a known follow-up will be analyzed.

Adolescent↗
PubMed2006

Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.

Base Sequence↗
PubMed2006

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR.

BACKGROUND: In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. RESULTS: The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. CONCLUSION: The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample.

Algorithms↗
PubMedDate not supplied

[Quantitative assay of 5-HT(1A) serotonin receptor gene expression in the brain].

Serotonin 5-HT(1A) receptors participate in the regulation of many kinds of behavior and are implicated in the mechanism of action of anxiolitics and antidepressants. The investigation of 5-HT(1A) receptor gene expression is complicated by low concentration of the receptor mRNA. Our method of quantification of the receptor gene expression in brain structures includes estimation of the concentration of genomic DNA contamination, the number of cDNA copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)--one of the "housekeeping genes", and the number of cDNA copies of 5-HT(1A) receptor in the sample. To evaluate the number of cDNA copies of the receptor and GAPDH, the fluorescence intensity of PCR-product was calibrated using genomic DNA-standard of a known concentration. The intensity of 5-HT(1A) receptor gene expression was corrected by genomic DNA contamination and was evaluated as a number of copies of 5-HT(1A) receptor cDNA per 100 copies of GAPDH cDNA. Using this method an increase of 5-HT(1A) receptor gene expression in the frontal cortex and amygdala in monoamine oxidase A knockout mice was shown.

Amygdala↗
PubMedDate not supplied

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture.

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.

Adsorption↗
PubMed2006

Validity of messenger RNA expression analyses of human saliva.

PURPOSE: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. EXPERIMENTAL DESIGN: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase ("no-RT") to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA. RESULTS: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated "RNA" extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, beta-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non-RNA-specific primers on saliva samples yielded PCR products for 36B4, beta-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the "no-RT" and "+RT" conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva. CONCLUSIONS: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.

Actins↗