Analyzing Microbes: Manual of Molecular Biology Techniques

365 Pages·2013·11.53 MB·English

by  Sukumar Mesapogu

Springer Protocols
.
Dilip Kumar Arora
Surajit Das
Mesapogu Sukumar
Editors
Analyzing Microbes
Manual of Molecular Biology
Techniques
Editors
DilipKumarArora
NationalBureauofAgriculturally
ImportantMicroorganisms
MaunathBhanjan
UttarPradesh
India
SurajitDas
DepartmentofLifeScience
NationalInstituteofTechnology
Rourkela
Odisha
India
MesapoguSukumar
NationalBureauofAgriculturally
ImportantMicroorganisms
MaunathBhanjan
UttarPradesh
India
ISSN1949-2448 ISSN1949-2456(electronic)
ISBN978-3-642-34409-1 ISBN978-3-642-34410-7(eBook)
DOI10.1007/978-3-642-34410-7
SpringerHeidelbergNewYorkDordrechtLondon
LibraryofCongressControlNumber:2013931210
#Springer-VerlagBerlinHeidelberg2013
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Preface
Thedisciplinemicrobiologyisresearchedactively,andthefieldisadvancing
continually.Itisestimatedthatonlyabout1%ofallofthemicrobespecies
on earth have been studied. Rapid advances in molecular biology have
revolutionizedthestudyofmicroorganismsintheenvironmentandimproved
ourunderstandingofthecomposition,phylogeny,andphysiologyofmicro-
bialcommunities.Theadventofmolecularbiologyhasofferedanumberof
revolutionary new insights into the detection and enumeration of soilborne
microorganisms. DNA sequences provide information on identifying
unknownspeciesfrom16SandITSrRNAsequencesofindividualbacterial
and fungal species. Molecular methods monitor both pathogens and also
beneficial organisms in soils for detection and quantification. The in-depth
exploitation of PCR potential led to more sophisticated variants of the
technique (improved even from the currently expanding real-time PCR)
thatincreasesthe speedandsensitivityinmicrobialidentification anddiag-
nostics. These molecular techniques provide new insights about their func-
tionsandinteractionswithinecologicalniches.
Analyzing Microbes—Manual of Molecular Biology Techniques is a
practicalguidetotheapplicationofimportantmolecularbiologytechniques
inmicrobiologicalresearch.Thechapters arewrittenbyagroup ofinterna-
tional scientists who are recognized authorities in their research areas from
universities/researchers and often the new techniques that are described.
These volumes are aimed for graduate, postgraduate, Ph.D. students, and
laboratory technicians working in different biotechnology/microbiology
laboratories. It is also valuable to the larger community of researchers who
haverecognizedthepotentialofgenomicsresearchandmaybebeginningto
explore the technologies involved.Moreover, the volumes are also targeted
ashandoutsforstudents,teachers,andresearchersworldover.
Thecentralpartsofthechaptersaretheexperimentalprotocolswhichare
presentedsoastobereadilyusedatthelaboratorybench.Althoughanumber
oftheproceduresdescribedrepresentthetriedandtrusted,wehavestrivento
include variants on existing technologies that an experiment can be per-
formed. These step-by-step protocols are intended to be concise and easy
to follow. Suggestions to successfully apply the procedures are included,
along with recommended materials and suppliers. A special feature of the
chaptersisthat,inadditiontotheprotocols,importantbackgroundinforma-
tionandrepresentativeresultsofapplyingthemethodsaregiven.References
are provided to enable the investigator to become better acquainted with
v
vi Preface
the topic. Researchers in any field that utilizes microbial systems will find
this work of value. In addition to microbiology and bacteriology, this book
highlights the current state-of-the-art molecular microbiology techniques in
biotechnology,microbiologyresearch,andenvironmentalmicrobiology.
TheaimofthebookAnalyzingMicrobes—ManualofMolecularBiology
Techniques has been to produce a self-contained laboratory manual which
willbeusefultobothexperiencedpractitionersandbeginnersinthefield.We
hope that this book stimulates yourcreativity and wish you success inyour
experiments.
MaunathBhanjan,UttarPradesh,India DilipKumarArora
Rourkela,Odisha,India SurajitDas
MaunathBhanjan,UttarPradesh,India MesapoguSukumar
Contents
1 MicrobialDNAExtraction,Purification,andQuantitation. . . 1
SukumarMesapogu,ChandraMouleswararaoJillepalli,
andDilipK.Arora
2 Fluorescent-BasedDetection,Quantitation,andExpression
ofViralGenebyqRT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . 17
ShellyPraveenandVikasKoundal
3 RestrictionEnzymesandTheirRoleinMicrobiology. . . . . . 31
SukumarMesapogu,ChandraMouleswararaoJillepalli,
andDilipK.Arora
4 GeneticFingerprintingTechniquesforMolecular
CharacterisationofMicrobes. . . . .. . . .. . . . .. . . .. . . . .. . 37
AnnetteReinekeandK.UmaDevi
5 AgaroseGelElectrophoresisandPolyacrylamideGel
Electrophoresis:MethodsandPrinciples. . . . . . . . . . . . . . . . 73
SukumarMesapogu,ChandraMouleswararaoJillepalli,
andDilipK.Arora
6 MolecularIdentificationofMicrobes:I.Macrophomina
phaseolina. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
BandamaravuriKishoreBabu,T.KiranBabu,andRajanSharma
7 MolecularIdentificationofMicrobes:II.Bacillus. . . . . . . . . 99
AnilKumarSaxena
8 MolecularIdentificationofMicrobes:III.Pseudomonas. . . . 105
BhimPratapSinghandRatulSaikia
9 MolecularIdentificationofMicrobes:IV.Vibrio. . . . . . . . . . 113
HirakRanjanDash,NeelamMangwani,andSurajitDas
10 MolecularIdentificationofMicrobes:V.methylotrophs. . . . . 123
KamleshK.Meena,ManishKumar,andD.P.Singh
11 PreservationandMaintenanceofMicrobialCultures. . . . . . 135
SudheerKumar,PremL.Kashyap,RuchiSingh,
andAlokK.Srivastava
vii
viii Contents
12 MicrobesfromExtremeEnvironment:Molecular
IdentificationProcedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
SurajitDas
13 ELISA-BasedIdentificationandDetectionofMicrobes. . . . . 169
JyotiVerma,SangeetaSaxena,andSunilG.Babu
14 AnalysisofMicrobialDiversityandConstruction
ofMetagenomicLibrary. . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
ThangamaniRajesh,JeyaprakashRajendhran,
andParamasamyGunasekaran
15 BioinformaticsToolsforInterpretationofDataUsed
inMolecularIdentification. . . . . .. . . . . . . . .. . . . . . . .. . . . 209
SuchiSmita,KrishnaP.Singh,BashirA.Akhoon,ShishirK.Gupta,
andShailendraK.Gupta
16 MolecularPhylogeneticsofMicrobes. . . . .. . . .. . . .. . . .. . 245
SurajitDasandHirakRanjanDash
17 MicroarrayTechnology:BasicConcept,Protocols,
andApplications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
P.P.DubeyandDineshKumar
18 MicroarrayAnalysisofDifferentFunctionalGenes
ofMicroorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
HirakRanjanDashandSurajitDas
19 DNACloningandSequencing. . . . . . . . . . . . . . . . . . . . . . . . 291
BandamaravuriKishoreBabu,AnuSharma,andHariKishanSudini
20 BiologicalSequenceAnalysis:AlgorithmsandStatistical
Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
SuchiSmita,KrishnaP.Singh,BashirA.Akhoon,
andShailendraK.Gupta
Appendix1:Biosafety,GLP,andBiosecurity. . . . . . . . . . . . . . . . 335
Appendix2:AddressforInstrumentsandChemicalsSuppliers. . . 339
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Contributors
Bashir A. Akhoon CSIR-Indian Institute of Toxicology Research,
Lucknow,India
Dilip K. Arora National Bureau of Agriculturally Important Microorgan-
isms,Kusumaur,MaunathBhanjan,UttarPradesh,India
Sunil G. Babu Department of Biotechnology, School of Bioscience and
Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar
Pradesh,India
Bandamaravuri Kishore Babu Cereals Pathology, International Crops
Research Institute for the Semi-Arid Tropics, Patancheru, Andhra Pradesh,
India
SurajitDas DepartmentofLifeScience,NationalInstituteofTechnology,
Rourkela,Odisha,India
Hirak Ranjan Dash Department of Life Science, National Institute of
Technology,Rourkela,Odisha,India
P.P. Dubey Department of Animal Genetics and Breeding, Guru Angad
DevUniversityofVeterinary,AnimalSciences,Ludhiana,Punjab,India
Paramasamy Gunasekaran Department of Genetics, Centre for Excel-
lenceinGenomicSciences,SchoolofBiologicalSciences,MaduraiKamaraj
University,Madurai,TamilNadu,India
ShishirK.Gupta SocietyforBiologicalResearchandRuralDevelopment,
Lucknow,India
Shailendra K. Gupta CSIR-Indian Institute of Toxicology Research,
Lucknow,India
Chandra Mouleswararao Jillepalli Department of Chemistry, Kakatiya
University,Warangal,AndhraPradesh,India
Prem L. Kashyap National Bureau of Agriculturally Important Micro-
organisms,Kusumaur,MaunathBhanjan,UttarPradesh,India
ix