PCR methods and applications最新文献

PCR is commonly used for mRNA quantitation. Previously described procedures are applied to one or a few specific mRNA sequences. We show here that methods used for amplifying heterogeneous cDNA populations can be applied to the quantitation of many mRNA species. This quantitation is achieved by dot blotting and hybridization with the corresponding probes after amplifying a bulk mRNA population. Only a single, two-round-amplification assay is required for quantitation of a whole set of mRNA species. The proportionality of input molecules to output signal was shown by performing a series of control experiments. We applied this technique to measure the relative variations of the MBP, Po, and MAG mRNA sequences in the normal trembler mouse model. The results were consistent with previously described Northern blot data. This quantitative PCR method provides a rapid and reliable way to quantify relative amounts of mRNA species in small amounts of total RNA by using internal controls.

Valid comparisons of gene promoter activities between different cell lines, and within a cell line, critically depend on accurate measurements of the number of genes introduced into the nuclei of cells. We have developed a simple method that allows direct and accurate quantitation of transfected plasmid DNA in cultured cells. The transfected DNA present in nuclei is copurified with genomic DNA without using phenol/chloroform extractions. DNA is amplified by PCR, and the amount of transfected DNA is read directly from a standard curve. By using the procedures described in this report, we have studied the relative expression of the Escherichia coli beta-galactosidase gene, driven by the wild-type Mo-MuLV LTR, in Rat-1 fibroblasts, FBJ v-fos-transformed Rat-1 (1302), and a revertant of v-fos-transformant (EMS-1-19) cell lines. The relative levels of expression of the transgene at 22 hr post-transfection in these three cell lines were 1:4:1, respectively, and at 48 hr post-transfection the respective ratios were 1:10.6:4. These results have significant implications for the use of cotransfected internal control plasmids to normalize data from transient transfection experiments to study promoter activities among different cell lines.

Qualitative and quantitative HIV-1 DNA and RNA PCR assays are proving to be useful in the diagnosis of HIV-1 infection in infants and in assessing the in vivo antiviral activity of new therapies and regimens in clinical trials. The use of these standardized commercial assays in conjunction with an external quality assurance program has ensured that results from different laboratories are comparable. In addition, real-time proficiency monitoring has the potential to detect problems immediately before patient data are compromised.

We have developed a method by which a researcher can quickly alter the specificity of a trans hairpin ribozyme. Utilizing this PCR method, two oligonucleotides, and any target vector, new ribozyme template sequences can be generated without the synthesis of longer oligonucleotides. We have produced templates with altered specificity for both standard and modified (larger) ribozymes. After transcription, these ribozymes show specific cleavage activity with the new substrate beta-glucuronidase (GUS), and no activity against the original substrate (HIV-1, 5' leader sequence). Utilizing this technique, it is also possible to produce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical system for the assessment of trans ribozyme activity.

To quantify angiotensin-converting enzyme (ACE) mRNA, we have developed a reverse transcription (RT)-coupled competitive PCR (RT-PCR) assay with a deletion mutant internal standard. The RT-PCR detects ACE mRNA from both human and bovine sources. ACE mRNA was detected in total RNA from cultured human saphenous vein smooth muscle cells (HuSV-SMCs) and from bovine pulmonary artery (BPA) SMCs. BPA-SMC expressed ninefold less ACE mRNA than BPA endothelial cells, and threefold less than HuSV-SMCs. Apparent amounts of ACE mRNA were 118,350 +/- 2,300 copies in HuSV-SMCs and 42,200 +/- 11,300 copies in BPA-SMCs per microgram of total cell RNA. The accuracy of the absolute values is subject to the limitations of the assumptions used to calculate them. These data support the hypothesis that components of the renin-angiotensin system are transcribed by SMCs.