molecular methods, pcr

1. Molecular MethodsMolecular Methods
in Diagnosis ofin Diagnosis of
Infectious DiseasesInfectious Diseases
PART 2PART 2

2. Diagnostic methods inDiagnostic methods in
MMicrobiologyicrobiology
 Task of the method – to make the
microorganism “visible” and
“measureable”
 Cultivation
 Bio-testing
 Immunological methods
 Biochemical methods
 Microscopy
 Molecular methods

3. Watson and CrickWatson and Crick ’s Discovery of’s Discovery of
DNA -Path to Molecular MedicineDNA -Path to Molecular Medicine

4. Genotyping MethodsGenotyping Methods
 Genetic methodsGenetic methods
generally seek togenerally seek to
detect polymorphisimdetect polymorphisim
at the level of nucleicat the level of nucleic
acidacid
 Genotypes are moreGenotypes are more
specific, more easilyspecific, more easily
quantified andquantified and
standardized amongstandardized among
the differentthe different
organismsorganisms

5. Basic requirementBasic requirement
 The genome isThe genome is
unique in eachunique in each
individualindividual
 UltimateUltimate
discriminatory stepdiscriminatory step
would be to sequencewould be to sequence
the entire genome ofthe entire genome of
every organism, butevery organism, but
not practical ornot practical or
economicaleconomical

6. What is PracticableWhat is Practicable
 Several methods inSeveral methods in
detecting nuclei aciddetecting nuclei acid
polymorphisim in apolymorphisim in a
chosen geneticchosen genetic
marker are commonlymarker are commonly
used to target theused to target the
genome or organism.genome or organism.

7. Kary Mullis - Nobel prize inKary Mullis - Nobel prize in
19931993
 Kary Mullis sharedKary Mullis shared
the 1993 Nobel Prizethe 1993 Nobel Prize
in Chemistry within Chemistry with
Michael Smith. MullisMichael Smith. Mullis
received the prize forreceived the prize for
his development ofhis development of
the Polymerase Chainthe Polymerase Chain
Reaction (PCR)Reaction (PCR)

8. PCR a Revolution inPCR a Revolution in
ScienceScience
 A process first describedA process first described
by Kjell Kleppe and 1968by Kjell Kleppe and 1968
Nobel laureate H. GobindNobel laureate H. Gobind
Khorana that allows theKhorana that allows the
amplification of specificamplification of specific
DNA sequences. TheDNA sequences. The
improvements providedimprovements provided
by Mullis have made PCRby Mullis have made PCR
a central technique ina central technique in
biochemistry andbiochemistry and
molecular biologymolecular biology

9. Polymerase ChainPolymerase Chain
ReactionReaction
Polymerase chain reactionPolymerase chain reaction ((PCRPCR) is a) is a
technique to amplify a single or few copiestechnique to amplify a single or few copies
of a piece of DNA across several orders ofof a piece of DNA across several orders of
magnitude, generating millions or moremagnitude, generating millions or more
copies of a particular DNA sequence. Thecopies of a particular DNA sequence. The
method relies on thermal cycling,method relies on thermal cycling,
consisting of cycles of repeated heatingconsisting of cycles of repeated heating
and cooling of the reaction for DNAand cooling of the reaction for DNA
melting and enzymatic replication of themelting and enzymatic replication of the
DNA.DNA.

10. Taq polymerase - backboneTaq polymerase - backbone
of PCR technologyof PCR technology
 Almost all PCRAlmost all PCR
applications employ aapplications employ a
heat-stable DNAheat-stable DNA
polymerase, such aspolymerase, such as
Taq polymeraseTaq polymerase ,,
an enzyme originallyan enzyme originally
isolated from theisolated from the
bacteriumbacterium ThermusThermus
aquaticusaquaticus..

11. Oligonucleotide – DNAOligonucleotide – DNA
PrimersPrimers
 This DNA polymeraseThis DNA polymerase
enzymatically assemblesenzymatically assembles
a new DNA strand froma new DNA strand from
DNA building blocks, theDNA building blocks, the
nucleotides, by usingnucleotides, by using
single-stranded DNA as asingle-stranded DNA as a
template and DNAtemplate and DNA
Oligonucleotide (alsoOligonucleotide (also
called DNA primers),called DNA primers),
which are required forwhich are required for
initiation of DNAinitiation of DNA
synthesis.synthesis.

13. Amplified sequences areAmplified sequences are
BlottedBlotted

14. PCR in ClinicalPCR in Clinical
MicrobiologyMicrobiology
 Molecular detectionMolecular detection
has mostly come tohas mostly come to
thethe clinicalclinical
microbiologymicrobiology
laboratory in the formlaboratory in the form
of PCR technology,of PCR technology,
initially involvinginitially involving
single round orsingle round or
nested proceduresnested procedures
with detection by gelwith detection by gel
electrophoresis.electrophoresis.

15. Helps Rapid DetectionHelps Rapid Detection
 Polymerase chainPolymerase chain
reaction (PCR)reaction (PCR)
techniques have led thetechniques have led the
way into this new era byway into this new era by
allowing rapid detectionallowing rapid detection
of microorganisms thatof microorganisms that
were previously difficult orwere previously difficult or
impossible to detect byimpossible to detect by
traditional microbiologicaltraditional microbiological
methods.methods.

16. Automation and MultiplexAutomation and Multiplex
PCRPCR
 With the advent ofWith the advent of
multiplex PCR, real-multiplex PCR, real-
time PCR andtime PCR and
improvements inimprovements in
efficiency throughefficiency through
automation, the costsautomation, the costs
of molecular methodsof molecular methods
are decreasing suchare decreasing such
that the role ofthat the role of
molecular methodsmolecular methods
will further increase.will further increase.

17. Progress in MolecularProgress in Molecular
MethodsMethods
 Molecular methodsMolecular methods
have now progressedhave now progressed
beyond identificationbeyond identification
to detect antimicrobialto detect antimicrobial
resistance genes andresistance genes and
provide public healthprovide public health
information such asinformation such as
strain characterisationstrain characterisation
byby genotyping.genotyping.

18. Molecular based TestsMolecular based Tests
needneed
 Nucleic acid-based tests used inNucleic acid-based tests used in
diagnosing infectious diseases usediagnosing infectious diseases use
standard methods for isolating nucleicstandard methods for isolating nucleic
acids from organisms and clinical materialacids from organisms and clinical material
and restriction endonulease enzymes, geland restriction endonulease enzymes, gel
electrophoresis, and nucleic acidelectrophoresis, and nucleic acid
hybridization techniques to analyze DNAhybridization techniques to analyze DNA
or RNAor RNA

19. Moleculardiagnostics is a set of methods
to study primary structure (sequence) of DNA
•Hybridization with complementary sequences
•Amplification (synthesis) of species specific sequences
PCR – polymerase chain reaction
-A-A-T-T-C-G-C-G-A-T-G-
- T-T-A-A-G-C-G-C-T-A-C-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-
-A-A-T-T-C-G-C-G-A-T-G-

20. Advances on PCR MethodsAdvances on PCR Methods
 Fairly recently, a newFairly recently, a new
method of PCRmethod of PCR
quantification hasquantification has
been invented. This isbeen invented. This is
calledcalled ““real-timereal-time
PCR”PCR” because itbecause it
allows the scientist toallows the scientist to
actually view theactually view the
increase in theincrease in the
amount of DNA as itamount of DNA as it
is amplified.is amplified.

21. REAL TIME ASSAYSREAL TIME ASSAYS

22. New Technologies – RealNew Technologies – Real
Time AssaysTime Assays
 The Real Time assays are proving to betterThe Real Time assays are proving to better
technologiestechnologies
1 Rapid1 Rapid
2 Quantitative measurement2 Quantitative measurement
3 Lower contamination rate3 Lower contamination rate
4 Higher sensitivity4 Higher sensitivity
5 Higher specificity5 Higher specificity
6 Easy standardization6 Easy standardization
Now a new gold standard for rapid diagnosis ofNow a new gold standard for rapid diagnosis of
virus infection in the acute phase samples.virus infection in the acute phase samples.

23. RT - PCRRT - PCR
 Proving to beProving to be
AccurateAccurate
PrecisePrecise
Easy to performEasy to perform
RT PCR technologiesRT PCR technologies
are easy to transferare easy to transfer
research Laboratoryresearch Laboratory
protocols toprotocols to
DiagnosticDiagnostic
LaboratoriesLaboratories

24. OVERVIEW of RT - PCROVERVIEW of RT - PCR
tissue
extract RNA
copy into cDNA
(reverse transciptase)
do real-time PCR
analyze results

25. Real Time ReportersReal Time Reporters
 All real time PCR systems rely upon theAll real time PCR systems rely upon the
detection and quantization of fluorescentdetection and quantization of fluorescent
reporter, the signal of which increases inreporter, the signal of which increases in
direct proportion of the amount of PCRdirect proportion of the amount of PCR
product in a reaction.product in a reaction.

26. REAL TIME PCRREAL TIME PCR
Cyber GreenCyber Green

USINGUSING
SYBER®SYBER®
GREENGREEN
 The simplest andThe simplest and
economical format theeconomical format the
reporter is the doublereporter is the double
strand DNA specificstrand DNA specific
dye SYBR ® Greendye SYBR ® Green
 Called as MolecularCalled as Molecular
Probe.Probe.

27. How SYBR Green worksHow SYBR Green works
 SYBR green binds toSYBR green binds to
double stranded DNAdouble stranded DNA
and upon excitationand upon excitation
emits lightemits light
 Thus as PCR productThus as PCR product
accumulates theaccumulates the
fluoresce increasesfluoresce increases

28. LimitationsLimitations ofof
SYBER®GreenSYBER®Green
AdvantagesAdvantages
InexpensiveInexpensive
Easy to UseEasy to Use
SensitiveSensitive
DisadvantagesDisadvantages
SYBR green will bindSYBR green will bind
to any doubleto any double
stranded DNA in astranded DNA in a
reaction, may result inreaction, may result in
an overestimation ofan overestimation of
the targetthe target
concentrationconcentration

29. Documentation ofDocumentation of
AmplificationAmplification
 The light emitted fromThe light emitted from
the dye in the excitedthe dye in the excited
state is received by astate is received by a
computer and shown oncomputer and shown on
a graph display, such asa graph display, such as
this, showing PCRthis, showing PCR
cycles on the X-axis andcycles on the X-axis and
a logarithmic indicationa logarithmic indication
of intensity on the Y-of intensity on the Y-
axis.axis.

30. Molecular Beacons
 Molecular Beacons
Uses FRET
Fluorescence Resonance Energy Transfer
Uses two sequence specific
 Oligonucleotide labelled with fluorescent
dyes

31. Molecular Beacons – RTMolecular Beacons – RT
PCRPCR
 Molecular beacons areMolecular beacons are
designed to adopt adesigned to adopt a
hairpin structure whilehairpin structure while
free in solution, briningfree in solution, brining
the fluorescent dye andthe fluorescent dye and
quencher in closequencher in close
proximity. When aproximity. When a
molecular beaconmolecular beacon
hybridizes to a target thehybridizes to a target the
fluorescent dye emitsfluorescent dye emits
light upon irradiation, andlight upon irradiation, and
rebind to target in everyrebind to target in every
cycle for signalcycle for signal
measurement.measurement.

32. Loop Mediated IsothermalLoop Mediated Isothermal
Amplification (LAMP)Amplification (LAMP)
 Loop mediated isothermal amplification isLoop mediated isothermal amplification is
a simple, rapid, specific and cost effectivea simple, rapid, specific and cost effective
nucleic acid amplification methodnucleic acid amplification method
characterized by use of 8 distinct regionscharacterized by use of 8 distinct regions
on the target gene.on the target gene.
 The amplification proceeds at a constantThe amplification proceeds at a constant
temperature using strand displacementtemperature using strand displacement
reaction.reaction.

33. LAMPLAMP
 Amplification andAmplification and
detection of gene can bedetection of gene can be
completed in a singlecompleted in a single
step, by incubating thestep, by incubating the
mixture of samples,mixture of samples,
primers DNA polymeraseprimers DNA polymerase
with strand displacementwith strand displacement
activity and substrates atactivity and substrates at
a constant temperature ofa constant temperature of
636300
c.c.

34. LAMPLAMP
 Compared with PCR, andCompared with PCR, and
real time PCR, the LAMPreal time PCR, the LAMP
has advantages ofhas advantages of
reaction simplicity andreaction simplicity and
detection sensitivity.detection sensitivity.
 The higher sensitivity andThe higher sensitivity and
specificity of LAMPspecificity of LAMP
reaction is attributed toreaction is attributed to
continuous amplificationcontinuous amplification
under isothermalunder isothermal
condition employing sixcondition employing six
primers recognizing eightprimers recognizing eight
distinct regions of thedistinct regions of the
target.target.

35. Advantages of LAMPAdvantages of LAMP
 LAMP functions on isothermalLAMP functions on isothermal
amplification.amplification.
 LAMP does not require any thermal cyclerLAMP does not require any thermal cycler
and thus can be performed even withand thus can be performed even with
water bath/heating blockwater bath/heating block
 LAMP method do not require sophisticatedLAMP method do not require sophisticated
temperature control devicestemperature control devices
 Cost effectiveCost effective

36. Lesser False Positives inLesser False Positives in
LAMPLAMP
 In LAMP bothIn LAMP both
amplification andamplification and
detection occurdetection occur
simultaneously during thesimultaneously during the
exponential phaseexponential phase
without going through thewithout going through the
plateau phase where theplateau phase where the
non spuriousnon spurious
amplification leads toamplification leads to
lower sensitivity and falselower sensitivity and false
positivity.positivity.

37. Loop Mediated IsothermalLoop Mediated Isothermal
Amplification in Clinical DiagnosisAmplification in Clinical Diagnosis
 LAMP technology proving to be ideal inLAMP technology proving to be ideal in
detection of DNA or RNA of the pathogenicdetection of DNA or RNA of the pathogenic
organismsorganisms
 Proving to be highly efficient in diagnosis of ViralProving to be highly efficient in diagnosis of Viral
and Bacterial infections,and Bacterial infections,
 LAMP is capable of detecting the presence ofLAMP is capable of detecting the presence of
pathogenic agents earlier than PCRpathogenic agents earlier than PCR

38. LAMP proving an efficientLAMP proving an efficient
TechnologyTechnology
 A one step single tubeA one step single tube
real time acceleratedreal time accelerated
reverse transcription loopreverse transcription loop
mediated isothermalmediated isothermal
amplificationamplification (RT-LAMP(RT-LAMP))
assays for rapid detectionassays for rapid detection
of some recentlyof some recently
emerged viral pathogenemerged viral pathogen
eg West Nile, SARS,eg West Nile, SARS,
Dengue, JapaneseDengue, Japanese
encephalitis Chikungunyaencephalitis Chikungunya
Norwalk, H5N1 highlyNorwalk, H5N1 highly
pathogenic avianpathogenic avian
influenza., andinfluenza., and
CMV,HPV,VZVCMV,HPV,VZV

39. Multiplex PCRMultiplex PCR
 TaqMan probes andTaqMan probes and
Molecular beacons allowMolecular beacons allow
multiple DNA species tomultiple DNA species to
be measured in the samebe measured in the same
sample ( Multiplex PCR)sample ( Multiplex PCR)
since fluorescent dyessince fluorescent dyes
with different emissionwith different emission
spectra may be attachedspectra may be attached
to different probesto different probes

40. Uses of Automated RT -Uses of Automated RT -
PCRPCR
 Several viral infections can be detected in acuteSeveral viral infections can be detected in acute
phase serum samples.phase serum samples.
 Increasingly used in for early and accurateIncreasingly used in for early and accurate
detection of almost all human viruses includingdetection of almost all human viruses including
Measles, Mumps, Herpes simplex viruses,Measles, Mumps, Herpes simplex viruses,
Rota viruses Noro virus, Influenzae virusRota viruses Noro virus, Influenzae virus
type A and B, Respiratory Synciticaltype A and B, Respiratory Syncitical
virus, SARS, Dengue Japanesevirus, SARS, Dengue Japanese
Encephalitis, Hepatitis B and C, WestEncephalitis, Hepatitis B and C, West
Nile, Chikungunya,HIV, Avian flu virus.Nile, Chikungunya,HIV, Avian flu virus.

41. Multiplex PCR in RealMultiplex PCR in Real
TimeTime
 Multiplex real timeMultiplex real time
quantitative RT-PCRquantitative RT-PCR
assays have beenassays have been
developed fordeveloped for
simultaneoussimultaneous
detectiondetection
identification andidentification and
quantification of HBV,quantification of HBV,
HCV and HIV-! InHCV and HIV-! In
plasma and Serumplasma and Serum
samples.samples.

42. Real-Time PCR MethodReal-Time PCR Method
Molecular BeaconsMolecular Beacons
 Molecular beaconsMolecular beacons
are short segments ofare short segments of
single-stranded DNAsingle-stranded DNA
(Figure 1). The(Figure 1). The
sequence of eachsequence of each
molecular beaconmolecular beacon
must be customizedmust be customized
to detect the PCRto detect the PCR
product of interest.product of interest.

43. StandardsStandards
 Same copy number in all cellsSame copy number in all cells
 Expressed in all cellsExpressed in all cells
 Medium copy number advantageousMedium copy number advantageous
– correction more accuratecorrection more accurate
 Reasonably large intronsReasonably large introns
 No pseudo geneNo pseudo gene
 No alternate splicing in region you wantNo alternate splicing in region you want
to PCRto PCR

44. Real-time PCRReal-time PCR
applicationsapplications
 Quantitation of gene expressionQuantitation of gene expression
 Pathogen detectionPathogen detection
 Viral quantitationViral quantitation
 Array verificationArray verification
 Drug therapy efficacyDrug therapy efficacy
 DNA damage measurementDNA damage measurement
 Quality control and assay validationQuality control and assay validation
 GenotypingGenotyping

45. Establishing PCRlaboratory
Sample handling
DNA preparation
Clean room
Stocksolutions
Laboratory
Mixing site
Thermocycler
Amplification
Detection
Documentation
QC & QA
Quality control & assurance
R & D
(Research and development)
Alternatives: - commercial kits
- robots + kits
No alternative

46. Emerging TechnologiesEmerging Technologies
in Molecular Diagnosisin Molecular Diagnosis

47. QIAGEN One Step RT-PCRQIAGEN One Step RT-PCR
KitKit
 The QIAGEN One StepThe QIAGEN One Step
RT-PCR Kit is designedRT-PCR Kit is designed
for easy and sensitivefor easy and sensitive
one-step RT-PCR usingone-step RT-PCR using
any RNA template. Aany RNA template. A
unique enzymeunique enzyme
combination and speciallycombination and specially
developed reaction bufferdeveloped reaction buffer
ensure efficient reverseensure efficient reverse
transcription and PCR intranscription and PCR in
one tube.one tube.

48. RT-PCR in one stepRT-PCR in one step
The Robus™ T I Kit is baseThe Robus™ T I Kit is base
 RobusT RT-PCRRobusT RT-PCR
Kits perform cDNAKits perform cDNA
synthesis andsynthesis and
PCR amplificationPCR amplification
of cDNAof cDNA
successively in asuccessively in a
single tube duringsingle tube during
a continuousa continuous
thermal cyclingthermal cycling

49. Uses and Advantages inUses and Advantages in
Testing by PCR MethodsTesting by PCR Methods
 Clinical diagnostics: detection and quantificationClinical diagnostics: detection and quantification
of infectious microorganisms, cancer cells andof infectious microorganisms, cancer cells and
genetic disordersgenetic disorders
 Capable of amplifying long targets, up to 6.0 kbCapable of amplifying long targets, up to 6.0 kb
 One-tube system allows rapid, sensitive andOne-tube system allows rapid, sensitive and
reproducible analysis of RNA with minimal risk ofreproducible analysis of RNA with minimal risk of
sample contaminationsample contamination
 Amplifies products from a wide variety of totalAmplifies products from a wide variety of total
RNA or mRNA sourcesRNA or mRNA sources

50. Advantages
Molecularmethods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&Dis absolutely necessary

51. Avoiding contaminationAvoiding contamination
 The single most important source of PCR
product contamination is the generation of
aerosols of PCR amplicons that is associated
with the post-PCR analysis. Methods for
eliminating this aerosol range from physical
design of laboratories and use of specific
pipettes to chemical and enzymatic approaches.
The choice of method is often dependent on the
frequency of amplification of a target amplicon
and the relative amounts and concentrations of
the amplicons created by the PCR.

52. LIGASE CHAIN REACTIONLIGASE CHAIN REACTION
 A method of DNA amplification similar to PCR.A method of DNA amplification similar to PCR.
 LCR amplifies the probe molecule rather thanLCR amplifies the probe molecule rather than
producing amplicon through polymerization ofproducing amplicon through polymerization of
nucleotides.nucleotides.
 Two probes are used per each DNA strand andTwo probes are used per each DNA strand and
are ligated together to form a single probe.are ligated together to form a single probe.
 LCR uses both a DNA polymerase enzyme andLCR uses both a DNA polymerase enzyme and
a DNA ligase enzyme to drive the reaction.a DNA ligase enzyme to drive the reaction.

53. STEPSSTEPS
 DenaturationDenaturation:: Heat double-strandedHeat double-stranded
DNA to denature it usually at 95DNA to denature it usually at 9500
C forC for
several minutes.several minutes.
 Annealing:Annealing: Annealing of probes toAnnealing of probes to
target DNA ( at 60target DNA ( at 6000
C).C).
 Ligation:Ligation: Joining of the probes byJoining of the probes by
thermostable DNA ligase. ( at 60thermostable DNA ligase. ( at 6000
C).C).

56. Nucleic acid sequence basedNucleic acid sequence based
amplification(NASBA)amplification(NASBA)
 Does not require thermal cycling.Does not require thermal cycling.

58. Strand DisplacementStrand Displacement
Amplification(SDA)Amplification(SDA)
 Two primer pairTwo primer pair
 Two enzymesTwo enzymes
 Modified deoxynucleoside triphosphateModified deoxynucleoside triphosphate