Hybridization with Nucleic Acid Probes: Part II: Probe Labeling and Hybridization Techniques

361 Pages·1993·14.16 MB·English

by  P. Tijssen (Eds.)

Hybridization with
nucleic acid probes
Part II: Probe labeling and
hybridization techniques
LABORATORY TECHNIQUES
IN BIOCHEMISTRY AND
MOLECULAR BIOLOGY
P.C. van der VLIET - Department for Physiological Chemistry,
University of Utrecht, Utrecht, The Netherlands
Volume 24
ELSEVIER
AMSTERDAM LONDON NEW YORK TOKYO
* * *
HYBRIDIZATION WITH
NUCLEIC ACID PROBES
Part 11: Probe labeling and
hybridization techniques
P. Tijssen
Institut Armand-Frappier
Uniuersite' de Que'bec
531, Boulevard des Prairies
ViNe de Laoal, Que'bec, Canada HNIB7
1993
ELSEVIER
AMSTERDAM . LONDON . NEW YORK . TOKYO
ELSEVIER SCIENCE PUBLISHERS B.V.
Sara Burgerhartstraat 25
P.O. Box 211, lo00 AE Amsterdam, The Netherlands
Library of Congress Cataloging-in-PublicationD ata
Tijssen, P.
Hybridization with nucleic acid probes / P. Tijssen.
-
v. (1-2). (Laboratory techniques in biochemistry and
molecular biology ; v. 24)
Includes bibliographical references and index.
--
Contents: pt. 1. Theory and nucleic acid probes pt. 2. Probe
labeling and hybridization techniques.
ISBN 0-444-89884-0 (v. 1. : acid-free paper). -- ISBN
0-444-89883-2 (v. 1. : acid-free paper : pbk.). -- ISBN
0-444-89886-7 (v. 2. : acid-free paper). -- ISBN 0-444-89885-9 (v.
2. : acid-free paper : pbk)
1. Nucleic acid probes. 2. Nucleic acid hybridization.
1. Title. 11 .Series.
QP519.E vol. 24
[QP624.5.D73]
574.19 ’2’ .28 S-dC2O 93-15 688
[574.87’328] CIP
ISBN 0 7204 4200 1 (series)
ISBN 0 444 89886 7 (hardbound Part 11)
ISBN 0 444 89885 9 (paperback Part 11)
ISBN 0 444 89888 3 (hardbound series Part I&II)
ISBN 0 444 89887 5 (paperback series Part I&II)
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Symbols used and their definition
BASH Basic aqueous hybridization solution
BCIP 5-Bromo-4-chloro-3-indolyplh osphate
BCR Branch capture reactions (strand displacement assays)
BFHS Basic formamide hybridization solution
BLOTTO Bovine lacto transfer technique optimizer (a blocking agent)
BM Boehringer Mannheim blocking agent
bP Base pairs (kbp, ‘kilo’ base pairs; kb, ‘kilo’ bases)
Ci Curie (radioactivity, 3.7 x 10’’ Bq)
DeS04 Dextran sulfate
dR/dC Sensitivity (change in response per unit concentration change)
s Decay constant (radioactivity) or discrimination ratio
(cross/specific hybridization)
CPm Counts per minute; equals dpm efficiency (of counting device)
dNTP Deoxynucleoside triphosphate (a,a s in [ a-32P]dATP, indicates
phosphate group closest to the sugar moiety, gamma is the
phosphate position farthest from the sugar)
dpm Disintegrations (nuclear transformations) per minute
a% Double-stranded
IAS Isothermal amplification system
ISH In situ hybridization
L Length of nucleic acid (shorter of the two annealing strands)
LCR Ligase chain reaction
1 Length of oligodeoxynucleotide
P Ionic strength ( = Eciz~c;i is ion concentration, zi its valence)
N Complexity of nucleic acid (sum of unique sequences in target
strand)
NSB Nonspecifically bound
OAc Acetate
PEG Polyethylene glycol
PCR Polymerase chain reaction (an enzymatic amplification method)
Qp-RS Qp replication system
RFE Rotating field electrophoresis
RGE Rotating gel electrophoresis
ss Single-stranded
sv Sievert (radiation dosage)
vi
t Time in seconds
Time required to hybridize half of the target nucleic acid; or, in
f0.5
the context of radioactivity, the half-life of a radioisotope
Temperature at which 50% of oligonucleotide duplexes are disso-
Td
ciated
Duplex retention temperature at which 50% of oligomer hybrids
Tdr
remain intact (during wash)
Ti Incubation temperature for annealing reaction
Tm (Melting) temperature at which 50% of duplex molecules become
dissociated
Experimentally observed melting temperature (oligomers)
Tm,obs
TAFE Transverse alternating field electrophoresis
Stacking temperature, indicative of stability of hybrids of two
succeeding bases
Contents
Symbols used and their definition ...................... V
Chapter 7. Labeling of probes and their detection .......... 269
7.1. Choice of label and labeling method .......................... 269
7.2. Radioisotopic labeling and detection of nucleic acids .............. 272
7.2.1. Principles of radioactivity and units .................... 272
7.2.2. Maximum and optimum radioactivity of probes ............ 274
7.2.3. Chemical stability of radiolabeled probes ................ 277
7.2.4. Choice of radiolabel ............................... 278
7.2.5. Detection of radioactive probes ....................... 280
7.2.5.1. Principles of autoradiography and use of films ..... 280
7.2.5.2. Autoradiography with nuclear track emulsion ..... 284
7.2.6. Safety considerations .............................. 286
7.3. Nonradioactive primary labels .............................. 287
7.3.1. Cherniluminescence ............................... 289
7.3.1.1. Acridinium ester probes ..................... 290
7.3.1.2. Acridinium labeling of probes ................ 293
7.3.2. Fluorochrome labels ............................... 293
7.3.2.1. Fluorochrome labeling of nucleic acid probes ..... 294
7.3.2.2. Fluorochrome labeling of streptavidin or antibody . . 296
7.3.2.3. Time-resolved fluorescence .................. 297
7.3.3. The use of enzymes as primary labels ................... 298
7.3.3.1. Labeling density of enzymes on probes .......... 298
7.3.3.2. Impact of solid phase on enzyme kinetics ........ 300
7.3.3.3. Characteristics of major enzymes .............. 302
7.3.3.3.1. Properties of alkaline phosphatase
(APase) ........................ 302
7.3.3.3.2. Properties of horseradish peroxidase
(POase) ....................... 303
7.3.3.3.3. Properties of P-galactosidase ........ 304
7.3.3.4. Colorimetric detection of enzymes ............. 305
7.3.3.4.1. Chromogenic detection of alkaline phos-
phatase ........................ 305
7.3.3.4.2. Chromogenic detection of horseradish
peroxidase 310
viii
7.3.3.5. Chemiluminescent enzyme substrates ........... 314
7.3.3.5.1. Dioxetane substrates for alkaline phos-
phatase and p-galactosidase ......... 314
7.3.3.5.2. Luminol substrates for peroxidase ..... 318
7.3.3.6. Labeling of nucleic acid with enzymes ........... 320
7.3.3.7. Conjugation of antibodies or streptavidin with en-
zymes .................................. 321
7.3.4. Electron-dense markers ............................ 321
7.4. Recognition systems ..................................... 321
7.4.1. Streptavidin and biotin-labeling of enzymes or linker proteins . 323
7.4.2. Antibodies ...................................... 325
7.4.2.1. Use of anti-nucleic acid antibodies ............. 325
7.4.2.2. Hapten antibodies ......................... 325
7.4.3. Sulfhydryl ligands ................................. 327
7.5. Overview of enzymatic incorporation of labels into probes .......... 329
7.6. Uniform incorporation of labels in nucleic acids ................. 330
7.6.1. Nick translation .................................. 330
..............
7.6.1.1. Radiolabeling by nick translation 334
7.6.1.2. Nick translation to incorporate secondary labels .... 335
7.6.2. Random-primed synthesis of labeled DNA ............... 337
7.6.2.1. Radiolabeling by random primer extension ....... 338
7.6.2.2. Incorporation of secondary labels by random primer
extension ............................... 340
7.6.2.3. Random-primed DNA synthesis by PCR ......... 341
7.6.3. Defined primer extension on single-strandedt emplates ...... 341
....
7.6.3.1. Upstream priming on single-stranded templates 342
7.6.3.2. Downstream priming on single-stranded templates . . 342
7.6.4. Labeling through sequential exonuclease-polymerase activities
(‘replacement synthesis’) ............................ 342
7.6.5. Preparation of nonradioactive probes by PCR ............. 347
7.6.6. In vitro synthesis and labeling of RNA by transcription ...... 349
7.6.7. Reverse transcription of RNA ........................ 353
7.7. End-labeling of probes ................................... 353
7.7.1. Labeling of 5’-ends of DNA or RNA ................... 353
7.7.2. Labeling of 3’ ends of DNA or RNA ................... 354
7.7.2.1. Radiolabeling of 3’-ends of DNA .............. 358
7.7.2.2. Nonradioactive labeling by tailing of 3’-ends of DNA 359
7.7.2.3. Labeling of 3’-ends of RNA .................. 359
7.8. Chemical modification of probes to introduce labels .............. 361
7.8.1. Introduction of reactive groups in nucleic acid by
transamination ................................... 362
7.8.2. Photohaptens (biotin, digoxigenin) ..................... 363
7.8.3. Psoralen crosslinkers .............................. 365
7.8.4. Crosslinking of haptenated or enzyme-labeled basic polymers . 366
ix
7.8.5. Radioactive labels ................................ 369
7.8.6. Mercurated nucleic acids for sulfhydryl-hapten ligands ...... 370
7.8.7. N-Acetoxy-N-2 acetylaminofluorene( AAF)l abeling ........ 371
7.9. Universal probes ........................................ 372
7.10. Biological probes ....................................... 373
Chapter 8. Mixed phase and solution hybridization formats 375
8.1. Materials and solutions ................................... 376
8.1.1. Solid phase systems ............................... 376
8.1.1.1. Membranes .............................. 377
8.1.1.2. Paramagnetic beads ........................ 381
8.1.1.3. Particulate solid phase ...................... 384
8.1.1.4. Microtiter plates .......................... 385
8.1.1.5. Affinity columns .......................... 386
8.1.1.6. Alternative solid phases ..................... 386
8.1.2. Equipment ...................................... 387
8.1.3. Solutions and buffers used in hybridization ............... 388
8.1.3.1. Blocking reagents ......................... 388
8.1.3.2. (Pre)hybridizations olutions .................. 391
8.2. Slot/dot blot hybridization on membranes ..................... 391
8.2.1. Immobilization of target nucleic acid ................... 393
8.2.2. Hybridization conditions ............................ 401
8.2.2.1. Prehybridization .......................... 401
8.2.2.2. Hybridization ............................ 403
8.2.2.3. Acceleration of hybridization by polymers ........ 406
8.2.2.4. Posthybridization washes .................... 407
8.2.3. Probe detection and analysis ......................... 410
8.2.4. ‘Fast’ blots of nucleic acid from cells ................... 413
8.2.5. Reprobing after stripping original probe from blots ......... 415
8.2.6. Hybridization to solid phase other than membranes ......... 416
8.3. (Semiksolution hybridization ............................... 416
8.3.1. Capture hybridization procedures ..................... 419
8.3.2. Reverse target capture hybridization ................... 421
8.3.3. Sandwich hybridization assays ........................ 426
8.3.3.1. Sandwich hybridization assays for DNA targets .... 427
8.3.3.2. Sandwich hybridization assays of RNA targets ..... 427
8.3.3.3. Sandwich hybridization followed by immunological
capture and detection ...................... 427
8.3.4. Solution hybridization procedures with label probe(s) only .... 329
8.3.4.1. Selective destruction of unhybridized probes ...... 329
8.3.4.2. Hybridization in concentrated solutions of chaotropes
or urea ................................. 430