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Technique / Molecular Biology / PCR
| The polymerase chain reaction (PCR) is commonly used in DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensics and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize for his work on PCR. |
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5' and 3' RACE (4)
Clony PCR (1)
Competitive PCR (1)
Degenerate PCR (1)
GC-rich PCR (2)
Genomic DNA PCR (1)
High fidelity long PCR (5)
In situ PCR (3)
Inverse PCR (2)
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Multiplex PCR (2)
Nested PCR (1)
Overlapping PCR (1)
PCR primer design (9)
PCR purification (3)
Quantitative real-time PCR (17)
RT-PCR (2)
Standard PCR (14)
Touchdown PCR (1)
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Application of Real-time Polymerase Chain Reaction RT-PCR (Technique review) (Hongbao Ma et.al., Michigan State University) Abstract: The real-time polymerase chain reaction (RT-PCR), also called quantitative real-time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction (kPCR), is a technique used to simultaneously quantify and amplify a DNA molecule. It is used to determine whether a specific DNA sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (qPCR), itself a modification of polymerase chain reaction (PCR). The procedure of RT-PCR follows the regular PCR procedure, but the DNA is quantified after each round of amplification. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. RT-PCR could be combined with reverse transcription polymerase chain reaction to quantify messenger RNA (mRNA) at a particular time for in a particular cell or tissue type. [The Journal of American Science. 2006;2(3):1-15]. (Reviews on PCR methods and techniques) 1: Rumsby G. An introduction to PCR techniques. Methods Mol Biol. 2006;324:75-89. Review. 2: Zhang C, Xu J, Ma W, Zheng W. PCR microfluidic devices for DNA amplification. Biotechnol Adv. 2006 May-Jun;24(3):243-84. Epub 2005 Dec 2. Review. 3: Kraytsberg Y, Khrapko K. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations. Expert Rev Mol Diagn. 2005 Sep;5(5):809-15. Review. 4: Adler M. Immuno-PCR as a clinical laboratory tool. Adv Clin Chem. 2005;39:239-92. Review. 5: Lauerman LH. Advances in PCR technology. Anim Health Res Rev. 2004 Dec;5(2):247-8. Review. 6: Niemeyer CM, Adler M, Wacker R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol. 2005 Apr;23(4):208-16. Review. 7: Ridgwell K. Genetics tools: PCR and sequencing. Vox Sang. 2004 Jul;87 Suppl1:6-12. Review. 8: Mike Makrigiorgos G. PCR-based detection of minority point mutations. Hum Mutat. 2004 May;23(5):406-12. Review. 9: Radstrom P, Knutsson R, Wolffs P, Lovenklev M, Lofstrom C. Pre-PCR processing: strategies to generate PCR-compatible samples. Mol Biotechnol. 2004 Feb;26(2):133-46. Review. 10: Kato K. Adaptor-tagged competitive PCR: study of the mammalian nervous system. C R Biol. 2003 Oct-Nov;326(10-11):941-7. Review. 11: Pohl G, Shih IeM. Principle and applications of digital PCR. Expert Rev Mol Diagn. 2004 Jan;4(1):41-7. Review. 12: Stirling D. Qualitative and quantitative PCR: a technical overview. Methods Mol Biol. 2003;226:181-4. Review. 13: Wendland J. PCR-based methods facilitate targeted gene manipulations and cloning procedures. Curr Genet. 2003 Nov;44(3):115-23. Epub 2003 Aug 19. Review. 14: Kricka LJ, Wilding P. Microchip PCR. Anal Bioanal Chem. 2003 Nov;377(5):820-5. Epub 2003 Aug 19. Review. 15: Dickerson SK, Papavasiliou FN. A modified digestion-circularization PCR (DC-PCR) approach to detect hypermutation-associated DNA double-strand breaks. Ann N Y Acad Sci. 2003 Apr;987:135-9. Review. 16: Hoorfar J, Cook N. Critical aspects of standardization of PCR. Methods Mol Biol. 2003;216:51-64. Review. 17: Sachse K. Specificity and performance of diagnostic PCR assays. Methods Mol Biol. 2003;216:3-29. Review. 18: Zhu J. Use of PCR in library screening. An overview. Methods Mol Biol. 2002;192:353-8. Review. 19: Hui EK, Wang PC, Lo SJ. PCR-based strategies to clone unknown DNA regions from known foreign integrants. An overview. Methods Mol Biol. 2002;192:249-74. Review. 20: Shen B. PCR approaches to DNA mutagenesis and recombination. An overview. Methods Mol Biol. 2002;192:167-74. Review. 21: Guo B, Bi Y. Cloning PCR products. An overview. Methods Mol Biol. 2002;192:111-9. Review. 22: Gustin K, Burk RD. PCR-directed linker scanning mutagenesis. Methods Mol Biol. 2000;130:85-90. Review. 23: Millican DS, Bird IM. Preparation of single-stranded antisense cDNA probes by asymmetric PCR. Methods Mol Biol. 1998;105:337-50. Review. 24: Schuler GD. Electronic PCR: bridging the gap between genome mapping and genome sequencing. Trends Biotechnol. 1998 Nov;16(11):456-9. Review. 25: Felix CA, Jones DH. Panhandle PCR: a technical advance to amplify MLL genomic translocation breakpoints. Leukemia. 1998 Jun;12(6):976-81. 26: Hooft van Huijsduijnen R. PCR-assisted cDNA cloning: a guided tour of the minefield. Biotechniques. 1998 Mar;24(3):390-2. Review. problems with housekeepings in real-time pcr (Forum) house-keeping gene for real-time PCR internal control 
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Related new papers and reviews
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Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism. Cytometry A. 2008 Feb;73(2):11-118
Authors: Fujiki Y, Tao K, Bianchi DW, Giel-Moloney M, Leiter AB, Johnson KL
Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P = 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs.
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