[Frontiers in Bioscience 2, c15-29, September 1, 1997
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IN SITU PCR. OVERVIEW OF PROCEDURES AND APPLICATIONS

Carlos A. Muro-Cacho, M.D., Ph.D.

Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of Medicine, Tampa, Florida, USA

Received 1/10/97 Accepted 7/15/97

TABLE OF CONTENTS


1. Abstract
2. Introduction
3. General considerations
3.1. Equipment
3.2 Technical aspects
3.3 Types of samples
3.4 Fixation
3.5 Proteolytic digestion
3.6 Additional pretreatments
4. Polymerase Chain Reaction (PCR)
4.1 Polymerases
4.2 Deoxynucleotide triphosphates (dNTPs)
4.3 Buffers
4.4 Primers
4.5 Cycling profile
5. In situ PCR Protocols
5.1 In situ RT-PCR
5.2 Direct In situ PCR
5.3 Indirect In situ PCR
6. Controls
6.1 Causes of false negativity
6.2 Causes of false positivity
6.3 DNA repair
6.4 Mispriming
6.5 Endogenous priming
6.6 Diffusion of PCR products
7. Applications
8. Acknowledgment
9. References
10. Appendices
Appendix 1 - General information
Appendix 2 - Sample preparation
Appendix 3 - Proteolytic digestion
Appendix 4 - Additional pretreatments
Appendix 5 - RT-PCR
Appendix 6 - Polymerase Chain Reaction
Appendix 7 - Direct In situ PCR
Appendix 8 - Indirect In situ PCR
Appendix 9 - Detection methods
11. Entire manuscript
Keywords: PCR, In situ PCR