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RT-PCR on Embryos Using Degenerate Oligonucleotide Primers

  • Anthony Graham
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

The polymerase chain reaction (PCR) is an incredibly versatile technique that has made a profound impact on many areas of biology. PCR is based on the use of oligonucleotides, which flank the region of DNA of interest and which are complementary to sequences on either DNA strand, to prime the replication of each strand by Taq DNA polymerase. This enzyme is thermostable, and consequently, one can go through cycles of template denaturation and renaturation after each round of replication and exponentionally amplify the sequence of interest. This procedure is very sensitive, and as such, it allows one to amplify, isolate, and utilize specific DNA sequences from vanishingly small quantities of starting material using either genomic or cDNA as a substrate.

PCR can also be used to facilitate the isolation of genes that are evolution-arily related, such as cognate genes from different species or multiple members of given gene families. In these cases, one is often trying to...

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Reaction Internal Primer Deoxynucleotide Triphosphate Avian Myeloblastosis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

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    Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989)Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y. 666Google Scholar
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    Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by guanindinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162, 156–159.CrossRefPubMedGoogle Scholar
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    Childs, S. R., Wrana, J. L., Arora, K., Attisano, L., O'Connor, M. B., and Massague, J. (1993) Identification of aDrosophila activin receptor.Proc. Natl. Acad. Sci. USA 90, 9475–9479.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Anthony Graham
    • 1
  1. 1.MRC Centre for Developmental NeurobiologyKing's College LondonLondonUK

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