PCR analysis

Classical approaches to plant DNA isolation aim to produce large quantities of highly purified DNA. However, smaller quantities of crudely extracted plant DNA are often acceptable for PCR analysis. Another efficient method for preparation of plant DNA for PCR is a single-step protocol that involves heating a small amount of plant tissue in a simple solution. Several factors influence nucleic acid release from tissue salt, EDTA, pH, incubation time and temperature. These factors must be optimized for different sample substrates. EDTA in the sample solution binds the Mg + cofactor required by the Taq polymerase in the PCR, so the EDTA concentration in the solution, or the Mg + concentration in the PCR, must be carefully optimized. 

Table 11 Primer sequences for PCR analysis of Roundup Ready (RR) Soy...

The PCR technique is very useful during all stages of the research and development of biotech crops. PCR analysis is used for gene discovery, event selection, screening, transformant identification, line selection and plant breeding. Quantitative real-time PCR is used to determine the number of transgene copies inserted in experimental... 

PCR analysis is one of the techniques used to generate data for the genetic analysis requirement. 

Aguila, C. D. P., C. G. Moura, H. Silva, A. J. D. Leitch, G. J. Moss, D. M. Wallace, S. Slemenda, S. B. Peiniazek, N. J. Wisvesvara, G. S. Ultrastructure, immunofluorescence, Western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS. J. Clin. Microbiol. 1998, 36, 1201-1208. 

Epe, C., Bienioschek, S., Rehbein, S. and Schnieder, T. (1995) Comparative RAPD-PCR analysis of lungworms (Dictyocaulidae) from fallow deer, cattle, sheep and horses. Journal of Veterinary Medicine B 42, 187-191. 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

The heat-induced retrieval protocol for extraction of formaldehyde-modified DNA from FFPE tissue sections provides a simple and effective method of DNA extraction from archival tissue samples.25,45 Based on PCR using three primer pairs ranging from 152-541 bp and a real time KTC-PCR analysis, the heat-induced retrieval protocol yields a better quality and quantity of DNA samples extracted from FFPE tissue sections than conventional methods of extraction.24 In addition, this heating protocol may provide an alternative approach for DNA extraction in some cases such as a recent publication by Ferrari et al. mentioned above.34... 

Cronin Breast cancer MasterPure TaqMan reactions RT-PCR analysis... 

Castiglione F, Degl Innocenti DR, Taddei A, et al. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues effects of the fixation on outcome reliability. Appl. Immunohistochem. Mol. Morphol. 2007 15 338-342. 

Figure 9. Cepheid GeneXpert for automated sample preparation and PCR analysis. (www.Cepheid.com March 18, 2004).

Gettemy JM, Ma B, Alic M, Gold MH (1998) Reverse transcription PCR analysis of the regulation on the manganese peroxidases gene family. Appl Environ Microbiol 64(2) 569-574... 

Levels of RNA (usually specific mRNAs) in a cell can be measured by well-established techniques such as northern blot analysis or by PCR analysis. However, the recent advent of DNA microarray technology has converted the identification and measurement of specific mRNAs (or... 

Reverse transcriptase. This enzyme is involved in the replication of retroviruses in vivo. It synthesizes a complementary DNA (cDNA) strand using RNA instead of DNA as its template. It is widely used to create a strand of cDNA from mRNA extracted from cells or tissue for cloning or for PCR analysis. 

Detection of chromosome fragments. Individuals with Turner s syndrome are, in some cases, mosaic for a portion of or for the entire Y chromosome (46,XY/45,X). Since such individuals may be at increased risk for gonadal tumors, Southern blot or PCR analysis has been used to detect the presence of Y chromosome segments in studies of DNA from peripheral blood samples. Similarly, the fetal sex as well as the presence of some aneuploid states (e.g., trisomy 18) can be determined by analysis of DNA from chorionic villi or amniotic fluid cells. 

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