PCR Lab

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AmericanSocietyofCytopathology
CoreCurriculuminMolecularBiology
Copyright2010AmericanSocietyofCytopathology
Broughttoyouby
AmericanSocietyofCytopathology
CoreCurriculuminMolecularBiology
Chapter4
LaboratoryOperations
DesigningMolecularLaboratoriestoDecrease
Contamination
KeishaN.Brooks,MS,CT,MB(ASCP)
UniversityofTennesseeHealthScienceCenter
Memphis,Tennessee
Brought
toyouby
Introduction
Foundationforthemolecularanalysisofspecimens
inmanylaboratoriesstartswiththepolymerase
chainreaction(PCR)
PCRAdvantage:Abilitytoamplifyverysmall
amountsofDNA
PCRDisadvantage:Processisvulnerableto
contamination
Laboratorydesignparamounttodecreasepossibility
ofspecimencontamination
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Real-Time PCR: Solution to

Contamination?
Providesdirectmeasurementofamplicon productasreactiontakes
place
AnalternativetotraditionalpostPCRanalysismethods;eliminates
needtohandlethesample
Selfcontainedautomatedsystem
NoneedtoopenPCRcontainers noamplicon escape
Eliminatestheneedforseparateprocessingrooms
Althoughadvantageous,contentsofindividualtubesmadeduringPCR
mayneedtobeanalyzedandcontainersmayhavetobeopened
*Willstillneedseparateroomforanalysis
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Contamination Issues
ImportantsourcesofPCRcontamination:
DevelopmentofPCRampliconaerosolsduring
postanalysis
Solutions:
Physicalseparationofthelaboratory
Useofaerosolproofpipettes
Targettemplate
RNAtemplateslessstablethanDNAtemplates;makes
RNAhighlysusceptibletoDNAcontamination
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Sources of Contamination
Previousamplificationandpurificationofplasmid
clones
Repeatedisolationoftemplatenucleicacids
Previouslyamplifiedmolecules(amplicons)
*ThecontrolandremovalofPCRampliconsisthebasis
foracontaminationcontrolprogram.
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PCR Activities
Samplepreparation
PCRreactionassembly
PCRexecution
PostPCRanalysis
*Theseactivitiescanbeclassifiedintwomajor
categories:PrePCRandPostPCR
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PCR Activities (Cont.)

PrePCRActivities
Isolationofnucleicacid
Assemblyofreactiontoamplifysamples
MustdetermineminimumneededequipmentforPCR
samplepreparation,reagentpreparation,andassayset
up
Risk:creatingaerosolsinpreparingRNAandDNAtemplates
Performunderhood
UVlightinhoodareapriortosamplepreparation
BenchtopsizedcabinetsequippedwithUVlight
PostPCR
PCRexecutionandanalysis
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toyouby
Contamination Control Program

SpaceandtimeseparationofpreandpostPCR
activities
Useofphysicalseparationaids
Useofultraviolet(UV)light
UseofaliquotedPCRreagents
Incorporationofnumerouspositiveandnegative
controlblankPCRs(watersubstitutedfornucleic
acidtemplate)
Useofoneormorevariouscontaminationcontrol
methods
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toyouby
Contamination Control
Twobroadmethodsofcontaminationcontrol:
PhysicalmethodstopreventdispersionofPCR
amplicons
Chemicalmethodsthatinactivatetheamplicons
abilitytobetemplatesinanewcycleofPCR
*MostsuccessfulPCRlabsuseaspectrumof
thesemethodstoeffectivelycontrol
contamination
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Contamination Control (cont)

PhysicalMethods
Positivedisplacement
Barrierpipettetipstopreventaerosols
Bothpreventthereintroductionofsmallamountsofacontaminating
aerosolizedsampleintothenextsamplethatispipetted
PipettetipsrecommendedinprePCRareasoflabwheresamplesarebeing
processedandtemplatenucleicacidsarebeingisolatedandpurified
TipsarenecessaryandcosteffectiveinprePCRlabbecausethereis
alreadyalargeamountofamplicon present
Adjuncttotipsistheuseoflaminarflowhoodorbiologicalsafetycabinet
topreparePCRsamplesandreagents
ReduceschanceofanexternalsourceofPCRamplicon contaminating
thesamplesandreagentsbeingmanipulatedforthesubsequentPCR
activities
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ChemicalMethods
UVPhotolinking
UsedinbothpreandpostPCRsetting
Fastreaction;effectivealsoforbiggeramplicons greaterthan700base
pairs
MostoftenusedinaprePCRsettinginwhichequipmentisinstalledina
smalltabletopcabinetandthenilluminatedbeforethePCRsare
assembled
Concerns:
UVlightexposure
Photoreactionfavorsthymidine overcytidine 10:1;amplicons A:T
richsequencesmoreeffienciently disabledthanA:Tpoorsequences
Decreasinglengthofamplicon usuallygivesalowerrateof
protection;shorteramplicons notwellcontrolled
Brought
toyouby

ChemicalMethods(cont)
UracilDNAGlycolase
EnzymealsoknownasUGD
HighlyeffectiveindestroyingPCRampliconswhenused
forsamplepreparation
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Space and Time Separation

Potentialcontaminationreductionbyseparatingthesourceof
amplicons
PostPCRandPrePCRrooms
Ifimpossible,designateareasforsamplepreparationand
PCRsetupthatareseparatefrompostPCRanalysisarea
EstablishdailyscheduleforperformingPCR
SamplepreparationandprePCRactivitiesshouldoccurin
themorning
PostPCRanalysisperformedinafternoon
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toyouby
Space and Time Separation (cont.)

Ifallactivitiesmustbeperformedinasingleroom:
Samplepreparationareashouldtakeplaceina
laminarflowhoodwithUVlight
Wallsofhoodwipedwithfreshlymadesolution
of10%bleachbeforeprocessingorprepping
samples
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toyouby

LaboratorySpaceArrangement
DeionizedwatershouldbepresentinpreandpostPCR
areas
Thereshouldbeseparatecentrifuges,storage
freezers/fridges,andstorageofsuppliesforbothareas
Phones,computers,andelectronicequipmentshouldnotbe
sharedbetweenthepre andpostPCRareas
*Itsimportanttonotethatrarelyislaboratoryspace
allocatedstrictlyforPCR;itsusuallysharedwithotherlab
procedures
PCRprotocolsmaybeintegratedintolaboperations
maximizesharingoffacilityandbenchspace
Brought
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PrePCRActivities
Isolationofnucleicacid
Assemblyofreactiontoamplifysamples
MustdetermineminimumneededequipmentforPCR
samplepreparation,reagentpreparation,andassay
setup
Risk:creatingaerosolsinpreparingRNAandDNA
templates
Performunderhood
UVlightinhoodareapriortosamplepreparation
BenchtopsizedcabinetsequippedwithUVlight
Brought
toyouby

EnvironmentalConcerns
AirHandling
Airhandlers needtobeseparateandairpressureindividually
adjustedineachlab
PrePCRarea:slightlypositiveairpressurecomparedtoairin
connectinghallway
PostPCRarea:slightlyreducedpressuretopullairinfromthe
outsideandpreventescapeofamplicons
Airhandlersshouldbeconnectedtoseparateairducts
Eachmustleadtoaseparatelocationforexhaust
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Protective Clothing/Equipment
Eachtechnologistshouldhavelabcoatsdesignatedonlyfor:
Generalmolecularlab
PrePCR
PostPCR
Inextremecases,disposablegownsandbootiesmaybe
used
Adhesivepaperorstickmatatlabentrances
Preventstraceamountsofdustanddebrisfromentering
lab
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Reagent Sterilization
BecausePCRlabsperformsomemethodsthatrequiresterilereagents,
somemayneedtobeautoclaved
Mostcriticalreagent:water(H2O)
SterileUSPwatercanbeconvertedtoPCRwaterbyfilteringitthrough
two0.45micronnitrocellulosefilters
Filtershavehighbindingcapacityfornucleicacidsandproteins
IflabisinvolvedinamplifyingverysmallquantitiesofbacterialDNA,
USPwatershouldbeautoclavedseparatelyfromallotherreagents
beforefiltration
ReagentsandsoliditemsdestinedforprePCRlabshouldbeautoclaved
separatelyfromothersupplies
Brought
toyouby
Sample PCR Laboratory Design
BenchSpace TissueCulture BenchSpace

Incubator
Sink
Computer
Microbiology with
network
Slight
SafetyHood
connection
Slight
4C Gel
Positive negative Imaging
Fridge
20C Air Air System
Freezer Pressure 80C Thermocycler Pressure

Upright
Freezer
4CFridge
Thermocycler 20C
Sink Freezer
RealTime
Type1 UV
Gel
PCR
Centrifuge Electrophoresis Oven
H20 Spectro. Equipment Equipment
PrePCRLabPostPCRLab
FlowofsamplesforPCRanalysis
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Sources
Mifflin,T.(dateunknown).SettingupaPCR
Laboratory.RetrievedJuly10,2010,from
http://www.biosupplynet.com/pdf/01_PCR_Prim
er_p.5_14.pdf
Buckingham,L.,&Flaws,M.(2007).Molecular
Diagnostics:Fundamentals,Methods,&Clinical
Applications.Philadelphia:F.A.DavisCompany.

PCR Lab

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Broughttoyouby

Real-Time PCR: Solution to

PCR Activities (Cont.)

Contamination Control Program

Contamination Control (cont)

Contamination Control (cont)

Contamination Control (cont)

Space and Time Separation

Space and Time Separation (cont.)

Space and Time Separation (cont.)

Space and Time Separation (cont.)

Space and Time Separation (cont.)

Sample PCR Laboratory Design

BenchSpace TissueCulture BenchSpace

Freezer Pressure 80C Thermocycler Pressure

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