In-Situ Pcr Techniques

This book describes comprehensive step-by-step protocols for the delineation of genetic amplification and histological detection techniques. Each procedure has been tested and validated for its sensitivity, precision, and reproducibility, and the authors give advice on the design of primers for PCR applications and on optimizing these protocols for use with plant, insect, and prokaryotic cells.

Omar Bagasra is with Claflin University in Orangeburg, South Carolina, USA.

FOREWORD

xi

(4)

HAROLD W. LISCHNER, M.D.

PREFACE

xv

1. REVIEW OF THE PCR TECHNIQUE

1

(18)

Kary Mullis's Invention: The Polymerase Chain Reaction

1

(2)

The Mechanism of the Chain Reaction

3

(4)

Oligonucleotide Primers

3

(1)

Denaturation and Annealing

4

(1)

Extension

5

(1)

Second Thermal Cycle

5

(1)

Geometric Amplification

6

(1)

Reverse Transcription: Making DNA from RNA

7

(2)

How to Design Primers

9

(7)

Primers for DNA Targets

9

(1)

Primers for RNA Targets

10

(2)

Commercially Available Primer Pairs

12

(1)

Length of Desired Amplicon

13

(1)

Sources for Sequence Data and Computerized Design of Primers

14

(1)

Details on Primer-Probe Design for Computer-hating Luddites

15

(1)

Annealing Temperature for Primers and Probes; Touchdown Protocols

16

(3)

2. PRELIMINARY SOLUTION-BASED REACTIONS

19

(10)

Extraction of DNA/RNA Using Commercial Preparations

20

(1)

Protocols

20

(2)

Extraction of DNA/RNA from Various Cell Suspensions, Including Mononuclear Cells, Other Blood Cells, and Single-cell Suspensions

20

(1)

Extraction of DNA from Paraffin-embedded Tissue

21

(1)

Extraction of DNA from Bone Fragments or Forensic Material (Example: Desiccated Tooth)

22

(1)

Solution-based PCR Procedures

22

(4)

Protocol: Solution-based DNA PCR

23

(2)

Adaptations for Solution-based RNA PCR

25

(1)

Verifying Probes and Detection Systems

26

(3)

Protocol: Dot-Blot Testing of Probes and Detection Systems

26

(2)

Southern Blot Alternative

28

(1)

3. PREPARATION OF GLASS SLIDES AND TISSUES

29

(8)

Glass Slides and Various Sealing Technologies

29

(1)

AES Silanation: Putting on the Positive Charge

30

(1)

Preparation of Tissue

31

(2)

Cell Suspensions

31

(1)

Cells Cultured on Slides

32

(1)

Paraffin-fixed Tissue

32

(1)

Plastic Sections

33

(1)

Frozen Sections

33

(1)

Protocols

34

(2)

Freezing the Tissue

34

(1)

Sectioning the Tissue

35

(1)

Archival Tissue

36

(1)

4. IN SITU PCR: DNA AND RNA TARGETS

37

(24)

Basic Preparation, All Protocols

37

(12)

Protocols

37

(5)

Heat-stabilization Treatment for DNA and RNA Messages

37

(1)

Fixation and Washes

38

(1)

Proteinase K Treatment

39

(1)

Alternative Method for the Proteinase K Treatment

40

(2)

Discussion of Optimizing Digestion

42

(1)

Discussion of DNAase Treatment for RNA Targets Exclusively

43

(2)

Protocols

45

(2)

Prepare a RNAase-free, DNAase Solution

45

(1)

Reverse Transcriptase Reaction

45

(2)

Discussion of RT Enzymes

47

(1)

Discussion of Primers for RT Reactions

47

(2)

PCR Amplification Procedures

49

(12)

Protocols

49

(5)

In Situ PCR with Conventional Sealing Technologies

49

(3)

Optimized In Situ PCR with Self-Seal Technology

52

(1)

In Situ PCR for One-step Reverse Transcription and PCR

53

(1)

Discussions

54

(7)

Attaching Cover Glasses with Nail Polish

54

(1)

Alternatives for Sealing Slides and Attaching Tissues

55

(1)

Hot Start Technique

56

(2)

Thermal Cyclers

58

(2)

Direct Incorporation of Nonradioactive Labeled Nucleotides

60

(1)

5. SPECIAL APPLICATIONS OF IN SITU AMPLIFICATION

61

(15)

Electron Microscopy

61

(1)

In Situ PCR and Immunohistochemistry

62

(2)

Cellular Antigens Resistant to Formalin Fixation

63

(1)

Labile Antigens Not Resistant to Formalin Fixation

63

(1)

Multiple Signals, Multiple Labels in Individual Cells

64

(1)

Recovery of Amplicons for Sequencing and Cloning

64

(2)

In Situ PCR and Gene Therapy Regimes

66

(2)

In Situ PCR on Chromosomes

68

(5)

Discussion of Uses

68

(1)

Protocol: In Situ PCR on Chromosome Spreads

69

(4)

Precautions

69

(1)

Initial Setup

70

(1)

Peripheral Blood

70

(1)

Cultured Cells

70

(1)

Arresting Cells at Mitotic Metaphase

71

(1)

Harvesting

71

(1)

Slide Preparation

72

(1)

In Situ PCR on Plant Tissues

73

(3)

Discussion of Uses

73

(1)

Protocol: In Situ PCR on Protoplasts

74

(2)

Liberating Protoplasts with Enzyme Digestion

74

(1)

Collecting Protoplasts and Rinsing Away Enzyme

75

(1)

Counting Cells and Slide Mounting

75

(1)

6. HYBRIDIZATION REACTIONS

76

(17)

Overview

76

(2)

Relative Merits Versus Other Techniques

78

(1)

Radioactive Probes Versus Nonradioactive Detection

79

(1)

The Empirical Arts of ISH

79

(1)

Hybridization after In Situ PCR

80

(4)

Probes

80

(1)

Design of the Oligo Probe

81

(1)

Time, Temperature, and Concentration

82

(1)

Labeling

82

(1)

Detection Methods

83

(1)

Controls for Hybridization

83

(1)

Labeling Oligonucleotide Probes

84

(2)

Protocols

85

(1)

(33) P Labeling of Probes

85

(1)

Nonradioactive Labeling-tailing with Dig-11-dUTP

85

(1)

Protocols: Hybridization Methods for In Situ PCR

86

(6)

General Hybridization

86

(2)

Posthybridization for (33)P Probe

88

(1)

Posthybridization for FITC or Other Fluorochrome-labeled Probes

88

(1)

Posthybridization for Peroxidase-based Color Development

89

(1)

Posthybridization for Alkaline-Phosphatase Color Development

90

(2)

Posthybridization for Digoxigenin-labeled Probe

92

(1)

Posthybridization of Directly Incorporated Labeled Nucleotides

92

(1)

7. VALIDATION AND CONTROLS

93

(3)

8. MATERIALS AND METHODS

96

(4)

Slides

96

(1)

Coplin Jars and Glass Staining Dishes

97

(1)

Solutions and Reagents

97

(3)

SELECTED BIBLIOGRAPHY

100

(9)

APPENDICES

109

(19)

1. Computer-assisted Designing of Primers and Probes

109

(10)

2. Amplification: The Detection of Rare Events

119

(9)

INDEX

128

(11)

ABOUT THE AUTHORS

139

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In-Situ Pcr Techniques

Summary

Author Biography

Table of Contents

Supplemental Materials

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In-Situ Pcr Techniques

Summary

Author Biography

Table of Contents

Supplemental Materials

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