APPENDIX

Safety in the molecular laboratory

Appendix

SAFETY IN THE MOLECULAR LABORATORY

Peter G. Isaac and Keith J. Edwards

Safety is a management issue. If you are working in a laboratory, or responsible for those working in a laboratory that uses any of the techniques listed in this book, you have a responsibility to understand the safety aspects of this work. There are many chem­icals and pieces of equipment listed in this book that can kill or maim if misused.

THE IMPORTANCE OF SAFETY TRAINING

Most incidents and accidents occur because someone was not trained adequately, or they were given a protocol without safety features marked. When accidents do occur, their effect can be minimized if the staff involved have been given adequate training. A golden rule is to make sure that there is, in each labor­atory, a person who is responsible for the safety supervision of the laboratory. Such a person is responsible for all the written records (incidents, accidents, details of safety training given, records of practice building evacuations) and for making sure that safety procedures are followed. It is important for this person to be given the authority to act as a safety supervisor in writing, and that they (and their immediate superiors) are given clear guidelines for administering safety issues (bear in mind that occasionally safety issues sometimes also become personnel or diSciplinary issues).

For every procedure, it is imperative to have written notes on how to handle the chemicals, how to cope with waste disposals and spills and a detailed protocol (specific to

your institution) for every procedure. Each procedure must be assessed for its likely haz­ards, and how waste products will be neu­tralized.

Further, a centralized database (either a card index or a computer database) should list all the chemicals used in the laboratory, where they are stored and information regarding safety issues (e.g. explosive in light, dispose of highly diluted in water, do not mix with nickel salts, etc.). Such a database should be consulted before a particular procedure is undertaken for the first time.

Every new employee or student should be given a set of the local laboratory rules (an example is given below). These rules explain in general terms the restrictions placed by working in a laboratory on behaviour, e.g. eating, drinking or applying cosmetics is not allowed in the laboratory. The local rules should also explain the rules to be followed in the event of fire. Should an emergency evacu­ation of the building be necessary the local rules should explain the procedure, plus the local rendezvous points.

The local laboratory rules should also give the name of the person who is responsible for safety management within the laboratory. Such a person is responsible for giving advice on safety matters, being aware of legal requirements of safety issues, keeping records of safety training, and keeping records of incidents and accidents.

It is important that new employees and students are given a copy of the local rules,

Molecular Tools for Screening Biodiversity. Edited by Angela Karp, Peter G. Isaac and David S. Ingram. Published in 1998 by Chapman & Hall, London. ISBN-13: 978-94-010-6496-5

488 Safety in the molecular laboratory

and their contents discussed with them on their first day of employment. This safety induction should also include a tour of the laboratory facility, indicating the location of fire extinguishers, fire exits, firefighting and other safety equipment (eyewash stations, etc.) and local quirks of the building that may give rise to special safety concerns (e.g. high voltage equipment rooms). Training is not a one-off occasion, and the safety induction should be followed up with a short meeting after three to four weeks to make sure that the new student or employee comprehends the rules, is following the rules, and to answer any queries that they may have. The date of the initial saftey training should be recorded, plus the date of the follow-up meeting, and the new student or employee should sign the records (under most legal systems - if you did not record the fact that you gave safety training, then it is assumed you did not give safety training). It is a management respons­ibility to make sure that the rules are under­stood, in particular with new employees whose first language is not that of the host laboratory. Especial care must be taken with such new colleagues, and it is a good idea, if at all possible, to have a fluent linguist in their native language to act as a translator during discussions of safety issues.

Laboratories must be equipped with eye­wash stations (and the bottles in these must be inspected at three-month intervals) and showers.

Incidents and accidents must be recorded separately - most laboratories have the for­mer at regular intervals, mercifully few have the latter. The difference between them is largely one of scale of the catastrophe. For instance, any safety failure that leads to some­one needing time off work is automatically registered as an accident (e.g. a severe cut hand requiring stitches). Incidents are trivial things that go wrong in a laboratory, that could have evolved into something nastier except for luck (a cut finger that requires only a plaster). Most insurers require to see the

incident and accident reports for a laboratory. The assumption is that if there are none, you have had plenty of accidents, you just didn't write them down. In all cases accidents and incidents must be recorded within 24 hours. The log of incidents and accidents becomes a management tool in safety administration as it allows frequent types of problems to be identified, and procedures put in place that prevent their reoccurrence.

SOME PARTICULAR HAZARDS MENTIONED IN THIS BOOK

The list below is not exhaustive, but just an attempt to highlight precautions that should be employed with some materials:

Ethidium bromide - this is a potent mutagen. It must not be inhaled - therefore wear a dust mask when handling a solid or opening a bottle containing ethidium bromide solution (solid crystallizes at the screw top). Adsorb to carbon before disposal or use commercial dis­posal system.

Acrylamide - a neurotoxin and carcinogen. Wear a dust mask. Treat unpolymerized acryl­amide with extreme caution.

Gel tanks - must be operated with a safety cover or interlock system. Users must be aware of the maximum voltage limits of gel tanks.

Ultraviolet light - can cause blindness and facial tumours. Wear a UV rated full face mask (goggles and chemical shields are insufficient).

Radiation - this book cannot give a full account of the procedures to deal with radio­isotopes, and the reader is advised to search for general texts.

As far as the UK is concerned, each institution must have a set of local rules for manipula­tion of isotopes that must be on permanent display (it is also a good idea to give a copy to

each new student and employee - and to get them to sign a register to say that they have read and understood the rules). Each labor­atory must also appoint a radiation protection supervisor (the RPS, responsible for the day­to-day management of radiation safety) and a radiation protection adviser (the RPA, often an independent consultant, responsible for independently reviewing radiation safety, and able to advise on changes in legislation etc.). Monitoring instruments must be reg­ularly calibrated. Adequate personal protec­tion must be provided (for the beta emitters like 32p that means perspex, not lead or glass). Personal dosimeters (body badges and finger tip monitors) must also be provided. In gen­eral, isotope sources should never be touched, but manipulated with remote handling tools. Radioisotopes, depending on their type and quantity, must be manipulated in either a 'supervised' area, or a 'controlled' area, and these areas must be marked as such. Facilities for the storage and disposal of radioisotopes must be provided.

AN EXAMPLE OF LOCAL LABORATORY RULES

In any laboratory, especially an unfamiliar laboratory, safety is of the utmost importance. This laboratory is no exception, therefore please take a few minutes to carefully read these notes.

Your safety supervisor is .................................. .

If they are not available contact ...................... '

or ........................................................................... .

1. Laboratory technique

a) Do not embark on a new or unfamiliar procedure until you understand what safety procedures are necessary (if in

An example of local laboratory rules 489

doubt, ask), and consult the chemicals database.

b) Good housekeeping is essential. Ensure benches, fume cupboards and floors are tidy and free of unnecessary equipment. Work in an orderly and organized man­ner.

c) Avoid skin contact with substances by tak­ing precautions to avoid spillage, by care­ful handling, and by wearing suitable gloves. Avoid transfer of contamination from gloves to other objects (e.g. tele­phones, door handles, refrigerators) by removing gloves beforehand. Wash hands regularly.

d) To avoid ingestion of substances by mouth:

• wear dust masks when weighing out chemicals

• do not pipette by mouth; • do not eat, smoke, drink, apply cos­

metics or lick labels in the labora­tory;

• do not store food or drink for human consumption in the laboratory.

e) Remove laboratory coats, gloves, and other protective equipment, and wash your hands, before leaving the laboratory.

£) Be conscious of the safety of cleaning and maintenance staff. Never leave chemicals or equipment in a dangerous condition. Dispose of broken glassware in appropri­ate clearly labelled containers, and rinse contaminated paper towels and tissues in copious amounts of water, or place in a sealed leak-proof polythene bag, before discarding them into waste bins.

2. Personal protective equipment

a) Laboratory coats must be worn over outer clothes and fastened at all times in the laboratory.

b) Eye protection must be worn at all times. c) Remove gloves to avoid transfer of con­

tamination to door handles, communal laboratory equipment, telephones, etc.

490 Safety in the molecular laboratory

3. Spillage and emergency procedure

a) Adequate equipment will be provided within the laboratory or in reasonable proximity to deal with chemical spillages. Deal with spills immediately they occur. In general, large spills of powders should be treated in a fashion that avoids raising a dust. If chemically appropriate, the chem­ical spill should be covered with a wet towel to prevent raising dust. A dust mask should be worn whilst gathering the chemical into the towel and disposing of it into a plastic sack. Report the spill to the laboratory superviser.

b) Where a spillage involves personal con­tamination:

• remove contaminated clothing; • wash with copious amounts of cold

water; • seek first-aid and/or medical advice

immediately; • report the contamination to the lab­

oratory supervisor.

Emergency showers and emergency eye wash facilities are available in laboratory areas. Make sure you know where they are located. c) Ensure that you are familiar with the loca­

tion, type and operation of fire extinguish­ers within the laboratory.

4. Evacuation

In the event of a building evacuation being necessary, if possible turn off all bunsen burn­ers or hazardous equipment, make sure that others within the laboratory have heard the alarm and are in the process of evacuation, proceed to the nearest fire exit and go to the rendezvous point outside the building. Give your name to the supervisor at the rendez­vous point, as well as details and location of any material or equipment left in a hazardous state, and names of colleagues who were in your laboratory but have not yet arrived at the rendezvous point.

AC repeats, defined, 288 acid citrate dextrose solution

(ACD),47 acrylamide, 154-5

deionization, 143 polyacrylamide gel, 138 safety, 488

Actinidia, RAPD analysis, 478-80 AFLP see amplified fragment length

polymorphism Africa, locust Schistocerca gregaria,

colonization studies, 423--4 agarose gel electrophoresis

detection of polymorphisms, 206-7

EtBr staining, 225 rucohol preservation, 47 algorithms

alignment, 334-6 branch swapping, 349 branch-and-bound, 348 construction of phylogenies from

discrete data, parsimony methods, 347-9

exhaustive search, 347-8 heuristic, 348 phylogenies, 347-9 rooting the tree, 349 stepwise addition, 348 tree islands, 349

alignment algorithm and problem, 334-6 ambiguity, DNA sequencing, 360 biological meaning, 334 coding sequences, ribosomal

DNA,272-4 local,336 multiple, 334-9

meaning of multiple alignment, 337-9

program~332,339 quadruple, 337 triple, 337

optimisation, 336 pairwise sequence alignment

problem, 334-5 progressive, 339

INDEX

alignment score, statistical sigificance, 337

allele-specific oligos (ASOs), 268 alleles

infinite alleles mutation model, 316,319

and level of differentiation, 303-6 Allium, rDNA amplification, 474 allozymes, 75-81 Alul buffer, 158-9 amplified fragment length

polymorphism (AFLP), 183-92

phylogenetic reconstruction, 345 reproducibility testing, European

laboratories, 191-2 schema, 184

AmpliTaq buffer, 113, 132 AmpliTaq DNA polymerase, 116 animru samples, preservation and

storage, 47-8 anion-exchange chromatography,

54-9,64-6 compared with silica gel-based

membranes, 54 Qiagen-tip-based methods, 54-9,

64-6 anti-Taq-polymerase antibody, 112 antibody conjugate development,

plastic binding, 96-7 anticoagulants, 47-8 ants, fungal symbiosis, small

subunit (ss) 16-18S rDNA, 271

aphids, DNA extraction, 27-9 Arabia, locust Schistocerca gregaria,

colonization studies, 423--4 Archaebacteria, 267 Auricularia spp., 5S RNAs, 338 automation, polymerase chain

reaction, 165-7 autoradiograms

analysis, 227 scoring, 196,201

autoradiographs, DNA sequencing, 332-3

Avena sterilis, 377

avidin, Vectrex-avidin, 282

Bacillus brevis, 5S RNAs, 338 bacteria, 5S RNAs, 338 Balaenoptera physalus (fin whale)

oligonudeotid primers, 252-3, 254 uni and biparental markers, 427

band-sharing coefficient, and individuality, 330-1

barley, 371-81 database searching, 240-8 dendrograms, 374, 468

parsimony analysis, 469 UPGMA analysis, 468

DNA extraction, 464-5 drought-induced genes, 467-9 genomes, 437-8 germplasm collections, 399 heat-shock probes, 465-7 molecular marker assays, 371-81

comparisons, 374-6 dendrograms, 374,468 fingerprinting, 372-3 genetic similarity, 373--4 genotypes used, 372

phylogeny, 437-40 RFLP markers, 229

beetles, colonization of Canary Islands, 419-21

binary data analysis band-sharing coefficient and

individuality, 330-1 co-segregation, 329 parentage analysis, 330-1 segregation, 329

biotin-streptavidin binding, DynaBeads, ssDNA, 160-1

blood samples, 8 blood storage, 47

avian blood, 106 reptilian blood, 106

bootstrapping, 339 branch swapping algorithms, 349 branch-and-bound algorithms, 348 Brassica oleracea, 377 5-brom0-4-chloro-3-indolyl

phosphate (BCIP), 127

492 Index

bromophenol blue, 19 buffers

AluI buffer, 158-9 AmpliTaq buffer, 113, 132 cetyl trimethyl ammonium

bromide (CTAB), 16 DNA extraction buffer (EB), 5-6,

10-11, 76 Dral buffer, 159 EB buffer, 5-6, 10-11, 76 electrode buffers, 77 enzymes, EC numbers, stains and

buffers, 77-8 G2 buffer, 55 gel buffers, 77 glucose-TElOO buffer, 27 HinfI, buffer for, 158-9 hybridization buffer, 138 loading buffer, 87 Na-TAE buffer see TAE buffer NET buffer, 16 QBT, QC, QF buffers, 56-7 Queen's lysis buffer, 47-8 RAPD buffer, 173 restriction endonuclease buffers,

158-9 RNase storage buffer, 11 sodium phosphate buffer, 284 5TEX buffer, 185 TAE buffer, 21, 87, 138, 141 Taq buffer, 197, 461 TBE buffer, 132, 173 TE buffer (pH 8.0), 6, 11, 16

Caenorhabditis elegans, 55 RNAs, 338 Camin-50kal parsimony,

phylogenies, optimality criteria, 346

Canary Islands, colonization by beetles, 419-21

case studies, 371-400 barley, 371-81 germplasm collections, 398-400 Lactuca, 388-93 potato, 371-81 tomato, 382-7 wheat, 394-7

cDNA, synthesis, 232-3, 234 cDNA probes, 232-6 cetyl trimethyl ammonium bromide

(CTAB),16 miniprep DNA extraction

protocol, 22-3 Chelex DNA extraction method for

PCR, preserved specimens, 42-4

chicken, 55 RNAs, 338 chloroform:isoamylaJcohol (CIAA),

30

chloroform extraction, PCR inhibitors, removal from DNA preparations, 43

chloroplast DNA (cpDNA), 223-8 55 RNAs, 338 amplification by conserved PCR

primers, table, 256 extraction, phenolics,

polysaccharides (ligneous species), 15-21

gymnosperms evolutionary evidence, 407-12 PCR-RFLP analysis, 403-6

oak genome analysis, 223-8 primers, consensus/universal,

256-64 Chorthippus parallelus (grasshopper)

colonization of Europe, 421-2 sequence data, 322

chromatographs, DNA sequencing, 333

Cicinde/a dorsa/is (tiger beetle), IT5 variation, 275

classification, 437-55 cleaved amplified polymorphic

sequence (CAPS), 262 clonal selection, 471 cloning

generation of codominant genetic markers, 217-22

RAPD product, sequence analysis, 221

recombinant DNA, screening, PCR,220

shot gun, 230, 231 cloning vector

isolation, 229-32 transformation, 231-2

codominant genetic markers, 217-22 colonization studies, insects, 419-25 cotton, rDNA, odd phylogenies,

275-6 cryopreservation, field collections,

47-8,49 CTAB buffer, 16 Cycas revo/uta, 55 RNAs, 338 cytochrome oxidase-l primers,

insects, 132

data analysis, 299-365 constructing phylogenies from

discrete data, maximum likelihood, 353-5

discrete data, 329-65 binary data, 329-31 gene trees vs species trees, 365 molecules vs morphology,

359-65 multiple alignment, 334-40

reading DNA sequences, 332-3 maximum likelihood methods,

359-65 measurement of genetic distance,

315-25 parsimony methods

RAPD,345 RFLPs, 345 sequence data, 344-6 VNTR,345-6

process partition, 362 software for PCs, 341-3

database searching, 239-48 Dellaporta method, plant DNA,

10-14,37 denaturing gradient gel

electrophoresis (DGGE), 135-43

dendrograms barley, 374, 468 Populus clones, 453 potato, 375 Rhododendron, 444 wheat, 396

Denhardt's solution, 92 deoxynucleotidyl transferase (TdT),

284 depurination, 90 dideoxy termination sequencing,

125 dideoxynucleoside triphosphates,

125 dideoxynucleotides

ssDNA sequencing, 160-1 terminator cycle sequencing kit,

122 differentiation

allelic level, 303-6 defined, 303 measure of polymorphism, 302-3

digoxigenin, 96 directed amplification of

minisatellite region DNA (DAMD),263

diversity measures see gene diversity

DM50-salt solution, preservation of field collections, 47

DNA extraction, 3-69 animal cells

difficult species, 27-31 total DNA, 5-9

anion-exchange chromatography, 54-9, 64-6

field collections, 46-8 organisms with high nuclease

activity, 29-31 for PCR sequencing, 122 plant cells

barley, 464-5 chloroplast DNA, high

phenolics, polysaccharides, 15-21

difficult species, 32-7 gymnosperms, 403-6 total DNA, 10-14

preserved specimens, 41-50 Qiagen Carlson protocol, 37 rapid, maxipreparations, 36-7 silica gel-based membranes, 54,

5'43,67-70 total DNA, 5-9 ultrapure DNA, 7-8

DNA extraction buffer (EB), 5-6, 10-11,76

DNA extraction medium, 33 DNA fingerprinting

blotting gels, capillary/vacuum, 103--4

defined, 101 electrophoresis, 103 hybridization, 105-6 multilocus (minisatellite

sequences), 101-8 VNTR sequences, 101-8

DNA fluorimetry, 106 DNA methylation, in vitro and in

vivo plant tissue cultures, 473 DNA precipitation, 227 DNA probes

hapten labelled, 96 hybridization, 92-4, 226-7 nonradioactive, 96-7 radiolabelling, 90-1

DNA quantification, 18-21 electrophoresis, 19-20 spectrophotometry, 20-1

DNA restriction enzyme reaction mixture, and gel electrophoresis, 86-9

DNA sequencing alignment ambiguity, 360 automatic sequencers, 127 autoradiographs, 332-3 by gel fractionation (nested sets),

125 by hybridization, 125 chromatographs,333 compared with PeR sequencing,

119 cycle sequencing, 127 denaturing gradient gel

electrophoresis (DGGE), 135-43

dideoxy termination sequencing, 125

dideoxynucleotides, ssDNA, 160-1

distance matrix, 335 fluorescent labelling, 126-7 manual sequencing, 127-8 multiple alignment, 334-9 pairwise sequence alignment

problem, 334-5 reading sequences, 332-3 routes, 125-30 temperature gradient gel

electrophoresis (TGGE), 135-43

see also alignment DNA thermal denaturation,

theoretical aspects, 144-6 Dollo parsimony, phylogenies,

optimality criteria, 346 DpnII,132 Dral buffer, 159 Drosophila, ITS variation, 275 drought-induced genes, barley,

467-9 Dryopteris acuminata, 5S RNAs, 338 DynaBeads, ssDNA, 160-3

EB buffer, 5-6, 10-11, 76 EBI, database searching, 240-1 EDTA (pH 8.0), 10, 18 electrode buffers, 77 EMBL database, 239 enzymatic sequencing, 125-6 enzymes

allozymes and isozymes, 75-81 EC numbers, stains and buffers,

77-8 Equisetum arvense, 5S RNAs, 338 Escherichia coli

RecA protein, triple-stranded DNA formation, 290

transformation with recombinant DNA,220

ethidium bromide, 88, 225 safety, 488

Eubacteria, 267 Europe, colonization by

grasshoppers, 421-2

field collections animal DNA extraction, 46-8 plant DNA extraction, 49-50

Fitch parsimony, phylogenies, optimality criteria, 346

flax cultivars, transposon activation, 474-5

fluorescent labelling, simple sequence repeats (SSRs), 208

fungi, 5S RNAs, 338

G2 buffer, 55 GC clamp, 147-8 gel buffers, 77

gel electrophoresis 2D,79-80

Index 493

denaturing gradient (DGGE), 141-3

DNA quantification, 19-20 high resolution, 206 isozyme analysis, 76-81 polyacrylamide, 79 sequencing gels vs agarose gels, 206 SSCP analysis, 153 temperature gradient (TGGE),

138-41 see also polyacrylamide gel

electrophoresis (PAGE) gel tanks, safety, 488 gene banks see germplasm

collections gene diversity

defined,304 inter / intralocus variance, 304 subdivision (differentiation), 303

gene diversity measures, 302-6 allelic level, 303-6 nucleotide level, 306-10 polymorphism index, 302 Shannon-Weaver information

function, 302 gene repression, and DNA

methylation, in vitro and in vivo plant tissue cultures, 473--4

gene trees, vs species trees, phylogenies, 365

GeneBank database, 239 mitochondrial genomes, 249

genetic distance coalescence time, 319 definition, 315 drift distances, 318-19 estimation

microsatellite data, 324-5 RFLP data, 323 sequence data, 322-3

geometric distances, 317-18 infinite allele models, 319 Jukes-Cantor model, 317, 321,

322,354 Kimura 2-parameter model, 317,

322,354 Mantel test, 321 measurement 315-25 mutation models

infinite alleles, 316-17, 319 nucleotide sequences, 316-17 stepwise mutation model, 203,

316,320 populations, equilibrium/ non­

equilibrium models, 317-21 sequences, 320-1

494 Index

genetic drift drift distances, 318-19 mutation models, 317

genetic markers, codominant, cloning, RAPDs, 217-22

genome plasticity, detection of hypervariable sequences, 471-84

genomes haploid, measures within/among

populations,305-6 somatic genome flux, 474-5

genomic DNA digestion and size selection,

280-2 probes, FU<LP markers, 229-36

germplasm, conservation, lettuce, 392

germplasm collections Hordeum, 399 screening, 398-400

gibberellic acid response, wheat, 474 glucose-TEl 00 buffer, 27 grasshopper Chorthippus parallelus

colonization of Europe, 421-2 sequence data, 322

guanidium (iso)thiocyanate, 232, 233

tissue preservation, 47 guanidium/hot phenol isolation

method, 232, 233 gymnosperms

HaeIII restriction patterns, 405 Norway spruce

library construction, 289-92 sequence variation, UTR, 137

Pinus halepensis, hypervariable cpDNA microsatellites, 407-12

populations, 403-17 species arranged (Strassburger),

404 various, PCR-FU<LP analysis of

cpDNA, 403-6

hairpin structures, 474 haploid genes, measures within/

among populations, 305-6 hapten labelled DNA probes, 96 HCI decontamination of laboratory

ware, 8 heat-shock probes, barley and

wheat, 465-7 Hennig86 program, 349, 350-1 Hin enzymes, 102-3 HinfI, buffer for, 158-9 homoplasy, causes, 360 Hordeum vulgare see barley horseradish peroxidase, 96

Hvhsp17, barley heat-shock probes, 465-7

hybridization, DNA fingerprinting, 105-6

hybridization apparatus, 107 hybridization buffer, 138 hypervariable sequences

screening, 471-84 in vitro and in vivo systems,

475--80

infinite alleles mutation model, 316, 319

insects ants, fungal symbiosis, small

subunit (ss) 16-18S rONA, 271

aphids, DNA extraction, 27-9 colonization studies, 419-25 cytochrome oxidase-I primers, 132 diversity using DNA sequences,

418-25 tiger beetle, ITS variation, 275

inter-simple sequence repeat amplification (ISSR), 263

intergenic spacer (IGS), 268 internal transcribed spacer (ITS)

sequences, 268, 270-1 invertebrates, DNA extraction, 27-9 IPTG,218 islands, Wright's model, 321 isoelectric focusing, 79 isozymes, 75--81

Jukes-Cantor model, genetic distance, 317, 321, 322, 354

Jungermannia subu/ata, 5S RNAs, 338

Kimura 2-parameter model, genetic distance, 317, 322, 354

Lactuca (lettuce) conservation of germplasm, 392 DNA sequencing, 391 molecular markers, 388-93

microsatellites, 390-1 RAPD,389 FU<LP, 229, 388-9 usefulness, 391-2

origins, 391 lambda cloning, Zap II, 290 lambda/HindIII DNA, molecular

weight markers, 102-3 LB medium, 218 lettuce see Lactuca library construction

Populus clones, 450-1 repeats, enrichment, 290-1

schema, 281 small size inserts (SSRs), 289-92

restriction and choice of enzyme, 289

size fractionation, 289-90 library screening

plant markers hybridization and washing

conditions, AC/GT and AG/ GT,292-3

pre-screening protocol (anchored PCR), 294-6

lichen symbiosis, small subunit (ss) 16-18S rONA, 271

loading buffer, 87 locust Schistocerca gregaria,

colonization studies, 423-4 Luria-Bertani medium, 218

MacVector, design of SSR primer pairs,287

maize, FU<LP markers, 229 Mantel test, genetic distance, 321 Megaptera novaeangliae (humpback

whale) mtDNA haplotype detection,

157-67 oligonucleotid primers, 252-3 parallel analysis of uni and

biparental markers, 426-30 Merlangius merlangus (whiting),

FU<LP survey, 323 microsatellite gels

pouring, 198-9 running and removal, 199-200 scoring, 196 visualizing, 200

microsatellites (SSRs), 195-211 AC repeats, 288 allele identification, 196 allele sizes, tomatoes, 210 animal markers, 279-87 biotin-tagged probes, 282 characteristics, 202-4 in conservation genetics, 197 data, genetic distance, 323 design of primer pairs using

MacVector, 287 estimation of genetic distance,

324-5 fluorescent labelling, 208 genotyping with microsatellite

markers, 195-205 humpback whale, 428 identification, 292-3 insects, 418-25 isolation and allelic variability,

202-3 Lactuca, 390-1 ligation and transformation, 284 linkage mapping, 203

loci, mutation rates, 203 Mus musculus (house mouse),

431-4 null alleles, 204 plant markers, 288-96

double-stranded polynucleotides, 293

library construction, 289-92 library screening, 292-3 oligonucleotides probes (AC)n,

293 PCR analysis of SSR

polymorphisms, 206-11 PCR reaction mixes, 209 primer design, 296 protocol for PCR pre-screening

of putatively positive clones, 294--6

sequencing of positive clones, 293-4

recombinants, percentage, 279 repeats, enrichment, 279-87

librarie~ schema,281 reproducibility testing, European

laboratories, 209-11 scoring, 196 screening and primer design, 284 sequence tagged microsatellite

site (STMS), 384 tomato, 383-4 wash conditions, selected probes,

283 see also libraries

minisatellite sequences, 101-8 directed amplification (DAMD),

263 discovery, 202 probes, single locus, 102, 106 VNTRs,202

Mismatch program, 146 mitochondrial DNA (mtDNA)

haplotype detection, Megaptera novaeangliae, humpback whale, 157-67

mtDNA primers, 249-64 Norway spruce, 413-17 nuclear copies of mt sequences,

251-3 oligonucleotide primers, 251 primers for animal mtDNA,

249-55 species list, 250

primers in plants, 256-64 conserved PCR primers, list,

260 molecular marker assays

barley, 371-81 inbreeding vs outbreeding, 371-81 potato, 371-81

molecular markers, hypervariable sequences, screening, in vitro and in vivo systems, 475-80

molecular weight markers, lambda/ HindIII DNA, 102-3

multiple alignment, 334-9 Clustal, 322, 339

Mus musculus (house mouse) origins, 432 partitioning of genetic diversity,

431-4 mutation models

genetic distance, 316-17 genetic drift, 317 in vitro rates, plant culture, 472-3 stepwise mutation model, 203,

316,320 mutation rates, plant tissue cultures,

472-4 mutations, point mutations,

detection by DGGE and TGGE,136

Na-TAE buffer see TAE buffer Nei's D, distance measure, infinite

allele models, 318, 319 NET buffer, 16 NET PVP Triton mixture, 16 NEXUS format, 350 nick-translation, 90-1 Nicotiana (tobacco)

autotrophy, 475 cpDNA, complete molecule, 256,

259 evolution, 478

Norway spruce see Picea abies nuclear DNA primers, 239-48 nuclease, DNA extraction from

organisms with high nuclease activity, 29-31

nucleic acids, thermal denaturation, theoretical aspects, 144--6

nucleotide sequences, mutation models of genetic distance, 316-17

nucleotides diversity and differentiation,

306-10 see also dideoxynucleotidesi

oligonucleotides

oak, genome analysis, chloroplast DNA,223-8

oligonucleotide primers, synthesis, 174

oligonucleotide probes, 268-9

pairwise sequence alignment problem, 334-5

see also alignment

Index 495

paraffin-embedded tissues for PCR analysis, 44-5

parentage analysis, 330-1 parsimony methods

construction of phylogenies from discrete data, 344-52

algorithms,347-9 optimality criteria, 346

PAUP program, 349 PCIA see phenol:chloroform:

isoamylalcohol Perkin-Elmer Gene Amp PCR

system, 123 Petunia cpDNA probes, 223-8 pGEM-T vector, 218, 221 phage lambda DNA, HindIII

digestion, 102-3 phenol:chloroform:isoamylalcohol,

12,34 phenol:chloroform extractions, 34,

123 phenol, buffered, 6, 28, 29-30 phenol extraction, PCR inhibitors,

removal from DNA preparations, 43

phenolics, polysaccharides (ligneous species), DNA extraction, 15-21

PHYLIP program, 349 phylogenies, 437-55

5s RNAs, 338 algorithms, 347-9 barley, 437-40 construction from discrete data

maximum likelihood, 353-5 parsimony methods, 344-52

gene trees vs species trees, 365 molecules vs morphology, 359-65 nuclear and organellar genomes,

362 optimality criteria, 346 ribosomal DNA, 275-6 software, 341-3,349-51 total evidence vs taxonomic

congruence, 359,360-3 tree topology, 361-3

Picea abies (Norway spruce) library construction, 289-92 mtDNA variation, 413-17 sequence variation, UTR, 137

Picea obovata (Siberian spruce), hybridization, 413

Pinus halepensis, hypervariable cpDNA microsatellites, 407-12

plant DNA cpDNA primers, 256-64 Dellaporta method, 10-14 difficult species, 27-31, 32-7

496 Index

plant DNA Grosshansdorf workshop, 36-7 plant tissue lysate, 56, 57-8

herbarium voucher specimens, 49 microsatellites (SSRs), 288-96

polymorphisms, PCR analysis, 206-11

miniprep procedures, 22-4 scaled-down CTAB, 22 for use in PCR, 23, 24

PCR applications, 32-7 silica gel dried plants, 49-50 somatic gene plasticity, 471 tissue cultures, mutation rates,

472-4 total DNA extraction, 10-14 see also chloroplast DNA;

microsatellites (SSRs); mitochondrial DNA (mtDNA)

plasmid DNA dephosphorylation, 229-30, 231 digestion with restriction

enzymes, 229, 231 point mutations, detection by

DGGE and TGGE, 136 Poland program, 144-6 poly(A) plus RNA, 232, 234 polyacrylamide gel, 138 polyacrylamide gel electrophoresis

(PAGE),185 polyethylene glycol (PEG) mixture,

123 polymerase chain reaction, 111-18

in AFLp, 185 amplification, 198 amplification mix, 138, 188

one biotinylated primer, 159-60 amplification protocols,

microsatellite markers, 195 annealing temperature, 117 applications, 111 arbitrary primed (AP-PCR) see

RAPDs automation, 165-7 Chelex DNA extraction method

for preserved specimens, 42-4

cleaved amplified polymorphic sequence (CAPS), 262

directed amplification of minisatellite region DNA (DAMD),263

heteroduplex formation, 139 influencing factors, 112 inhibitors, removal from DNA

preparations, 43 inter-simple sequence repeat

amplification (ISSR), 263

internal transcribed spacer (ITS) region, 112

isolation of microsatellite markers, animals, 282-3

isolation of total plant DNA, 23-4 MacVector, design of SSR primer

pairs, 287 magnesium concentration, 116 multiplex, 197 PCR-RFLPs, 131-4 primers, design, 284-5 process, schema, 114 psoralen crosslinking, 147-8 purification prior to restriction

digestion, 133 RAPDs, 171-9 reaction mixtures, 198,460-1

gymnosperms, 405 restriction digestion, 133-4 screening, recombinant DNA

clones, 220 sequence analysis of cloned

RAPD product, 221 sequence characterized amplified

regions (SCARS), 262 sequence-tagged microsatellite

site (STMS), 262 sequence-tagged site (STS), 262 sequencing, 119-30 single primer amplification

reaction (SPAR), 263 single-strand conformation

polymorphism (SSCP), 150-1, 152-6

see also DNA sequencing polymorphism

differentiation, 302-3 measures within/among

populations, 301-11 nucleotide level, measures

within/ among populations, 306-10

polysaccharides, plants (ligneous species), DNA extraction, 15-21,49

populations,403-34 differentiation, 302-3

calculated from restriction data, 309

distances between, 302-3 diversity, calculated from

restriction data, 308-9 genetic distance

equilibrium models, 321-2 non-equilibrium models, 317-21

gymnosperms, 403-17 haploid genes, measures within/

among populations, 305-6 humpback whale, 426-30

insects, 418-25 mouse, 432-4 polymorphism, measures within/

among populations, 301-11 Populus clones

dendrograms, 453 DNA isolation, 449 library preparation, 450-1 primers used in PCR, 450 RAPD fingerprinting, 449-55

analysis of clones, 176-9 potassium acetate solutions, 11 potato

molecular marker assays, 371-81 dendrograms, 375 fingerprinting, 372-3 genetic similarity, 373-4 genotypes used, 372 RAPD and AFLPs, 379

RFLP markers, 229 wild and cultivated, relationships,

378 preserved specimens

animal samples, 47-8 Chelex DNA extraction method

for PCR, 42-4 DNA extraction, paraffin­

embedded tissues for PCR analysis, 44-5

plant samples, 49-50 primers, 239-64

computer programs (Malign and Primer),255

cpDNA and mtDNA primers in plants, 25tH;4

design, 296 nuclear DNA primers, 239-48 for PCR, design, 284-5 primers for animal mtDNA,

249-55 ribosomal DNA, 267-76

probes, 217-36 design, 221 isolation, 217-36 labelling, 226 preparation, 226 ribosomal DNA, 267-76 see also microsatellites; primers;

VNTR probes and primers, simple repeats,

279-96 psoralens, crosslinking, 147-8

QBT, QC, QF buffers, 56-7 Qiagen Carlson protocol, DNA

extraction, 37 Qiagen-tip-based methods of DNA

extraction, 54-9 quadruple alignment, 337

Queen's lysis buffer, 47-8 Quercus spp., Petunia cpDNA

probes, 223-8

radiation, safety, 488--90 radiolabelling, 90-1

VNTR sequences, 104 random primed labelling, 90-1 randomly amplified polymorphic

DNAs (RAPDs), 171-9 cloning to generate codominant

genetic markers, 217-22 cpDNA of gymnosperms, 403--6 phylogenetic reconstruction, 345 polymorphism, 477-9 purification of product of interest,

219-20 reproducibility of profiles, 463 reproducibility testing, 176-9 screening for useful variation,

45%3 in vitro culture, 476-7 specificity levels, 477-9 troubleshooting, 462

storage of gels, 175 RAPD buffer, 173 rbel gene

barley, 438--9 dicots, sequencing primers, 258 trees, 365, 438

rbeS gene, thermal denaturation, 144-6

rDNA see ribosomal DNA recombinants, percentage of

microsatellites, 279 restriction endonuclease buffers,

158--9 restriction endonucleases, 87, 157,

158 plant cpDNA probes, 223 for plants, 289 preparation for DNA

fingerprinting, 102-3 Tsp5091, 289 see also Hin

restriction fragment length polymorphisms (RFLPs)

analysis, 85-97 basic principle, 86 DNA restriction and gel

electrophoresis, 86-9 hybridization, 92-4 kits, 96-7 PCR-RFLPs, 131-4 radiolabelling, 90-1 Southern blotting, 89-90 stress-responsive genes in

plants, 464-70 barley cultivars, 466

data, genetic distance, 323 genomic DNA probes, 229-36 phylogenetic reconstruction, 345

Rhododendron assessment criteria, 442 biodiversity measurement, 441-8 consensus tree, 445 dendrogram of cophenetic

correlations, 444 Hymenanthes project, 446 PCR ITS analysis, 112 RAPD,442 taxonomic conclusions, 444-6

ribosomal DNA alignment of coding sequences,

272-4 amplification, Allium, 474 analysis of data, 270 chromosomal organization and

homogenization, 275 concerted evolution, 269-70 expansion segments, 268 hybridization, 268--9 intergenic spacer (IGS), 268 internal transcribed spacer (ITS)

sequences, 268, 270-1 odd phylogenies, 275-6 organization, 267 probes and primers, 267-76 sequencing

coding regions, 269 spacers, 269

small subunit (ss) 16-185 rDNA, 267,271-4

smallest, 267 spacer length, 475 use, diversity studies, 269-74 ribulose-l,5-bisphosphate

carboxylase see rbel rice

cultivars, variability, 476, 478 germplasm screening, 398

RNA, 5s RNAs, phylogenies, 338 RNA, total, isolation see anion­

exchange chromatography; silica gel-based membranes

RNase A, 29 RNase solution, 12 RNase storage buffer, 11 rooting the tree algorithms, 349

safety,487-90 laboratory rules, 489-90 list of hazards, 488--90

SaulA/B linker sequences, 280, 283 Sehistoeerea gregaria (locust),

colonization studies, 423-4 screening

Index 497

genome plasticity, hypervariable sequences,471-84

germplasm collections, 398-400 library

plant markers hybridization and washing conditions, AC/GT

and AG/GT, 292-3 pre-screening protocol (anchored PCR), 294-6

PCR, recombinant DNA, cloning, 220

useful variation RAPD markers, 459--63 stress-responsive genes, RFLP

analysis, 464-70 screening, basic, 71-212

see also AFLP; DNA fingerprinting; isozymes;

microsatel1ites; PCRs; RAPDs; RFLP

SDS-hybridization solution, 224 seeds, RAPDs technique, 172-9 SeqNet, 239-40 sequence characterized amplified

regions (SCARS), 262 sequence-tagged microsatellite site

(STMS), 262, 384 sequence-tagged site (STS), 262 Shannon index, 467 Shannon-Weaver information

function, 302 short tandem repeats (STRs) see

microsatellites (SSRs) shot gun cloning, 230, 231 silica gel drying, 49-50 silica gel-based membranes, 54,

59--63, 67-70 compared with anion-exchange

chromatography, 54 DNeasy spin columns

animals, 67-8 plants, 61-3, 68-9

QIAamp spin columns animals, 60-1 plants, 59-60

silver staining, SSCP analysis, 153, 154

simple sequence repeats (SSRs) see microsatellites

single primer amplification reaction (SPAR),263

single-locus minisatellite probes, 106 single-strand conformation

polymorphism (SSCP), 150-1, 152-6

modifications, 164 SOB medium, 218 SOC medium, 219

498 Index

sodium acetate solutions, 11 sodium dodecyl sulphate (SDS), 11 sodium phosphate buffer, 284 software, phylogenies, 341-3,

349-51 software for PCs, 341-3, 349-51 Solanum tuberosum see potato somatic genome flux, 474--5 Southern blotting, 225--6

RFLP analysis, 89-90 Southern hybridization, tomato, 384 spectrophotometry, DNA

quantification, 20-1 SSCP see single-strand conformation

polymorphism ssDNA

DynaBeads M-280 streptavidin, 160-3

sequencing, dideoxynucleotides, 160-1

SSRs see simple sequence repeats stepwise mutation model, 203, 316,

320 STEX buffer, 185 streptavidin-DynaBeads

PCR, solid-phase sequencing, 254 ssDNA,16O

stress-responsive genes in plants, RFLP analysis, 464--70

T7 polymerase, 125 TAE buffer, 21, 87, 138, 141 TAE solution, 21 TaHSP16.9, wheat heat-shock

probes, 465-7 Tamura-Nei gamma genetic

distance model, 317 Taq buffer, 197,461 Taq polymerase, 174, 189, 218

AmpliTaq DNA polymerase, 116 anti-Taq-polymerase antibody, 112 oligonucleotide labeling, 208

TBE buffer, 132, 173 TE buffer (pH 8.0), 6, 11, 16 temperature gradient gel

electrophoresis (TGGE), 135-43

modifications, 147-8 single-strand conformation

polymorphism (SSCP), 150-1, 152-6

temporal, 149 terminal deoxynucleotidyl

transferase (TdT), 284 thermal denaturation, theoretical

aspects, 144--6 thermocycling,SSRs,209 tiger beetle, ITS variation, 275 tobacco see Nicotiana tomato

allele sizes, simple sequence repeats (SSRs), 210

cultivars, 382-7 molecular markers

comparisons, 385 microsatellites, 383-4 protein and DNA markers, 382 RAPDs, 383, 476-7 RFLP markers, 229, 382-3

transposons, activation, 474 Trapa natans (water chestnut), RAPD

analysis, 479 tree islands algorithms, 349 triple alignment, 337 triple helix DNA methods, 290 Tris (pH 8.0), 10 Triticae see barley; wheat Tsp5091, restriction endonuclease,

289

ultraviolet light, safety, 488

variable number tandem repeats (VNTRs)

DNA fingerprinting, 101-8

hypervariable tandem repeats, human genome, 202

labeling, 104 minisatellites, 202, 286 phylogenetic reconstruction, 345 simple sequence oligonucleotide

repeats, 286 variance

inter/intralocus,304 inter /intranucleotide, 307 sampling, decomposition at

diploid level, 305 variant richness

measures of polymorphism within/ among populations, 301-2

screening for, 459-64 variation; hypervariable sequences,

screening, in vitro and in vivo systems, 475-80

Vedttex-avidin,282

Wagner parsimony, phylogenies, optimality criteria, 346

whale see Balaenoptera physalus (fin whale); Megaptera novaeangliae (humpback whale)

wheat dendrogram~396 drought-induced genes, 467-9 gibberellic acid response, 474 heat-shock probes, 465-7 molecular markers, 394--7

AFLP, 186, 394--5 polymorphism, 186

RAPD,394 RFLP, 229

whiting Merlangius merlangus, RFLP survey, 323

X-Gal,218