Post on 07-May-2015
11
22
Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)
• A related, more recent Nobel Prize (Chemistry, 1993) was given for the Polymerase Chain Reaction (PCR) to Kary Mullis
• This puts together the concepts of DNA synthesis catalyzed by a polymerase, denaturation, and annealing.
"In Berkeley it drizzles in the winter. Avocados ripen at odd times and the tree in Fred's front yard was wet and sagging from a load of fruit. I was sagging as I walked out to my little silver Honda Civic, which never failed to start. Neither Fred, empty Becks bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome."
33
PCRAGAROSE GEL ELECTROPHORESIS
THE FINAL PRODUCT
3 TO 4 HOURS
U V VISUALIZATION
44
There are several reasons for the use of PCR:
1. Difficulties in identification of bacteria
2. Large time required for the identification with culture techniques (more than two days)3. The media required for the identification and confirmation of bacteria are very expensive4. A few bacteria in the environment are viable but not culturable.
55
POLYMERASE CHAIN REACTIONPOLYMERASE CHAIN REACTION
HAS MULTITUDE HAS MULTITUDE OF OF
APPLICATIONS APPLICATIONS IN IN
MICROBIOLOGYMICROBIOLOGY
66
DIAGNOSTIC APPLICATIONS OF DIAGNOSTIC APPLICATIONS OF PCRPCR
THERE ARE THREE PRIMARY THERE ARE THREE PRIMARY DIAGNOSTIC APPLICATIONS OF PCRDIAGNOSTIC APPLICATIONS OF PCR
Detecting pathogens using genome – Detecting pathogens using genome – specific primer pairs.specific primer pairs.
Screening specific genes for unknown Screening specific genes for unknown mutations.mutations.
Genotyping using known STS markers.Genotyping using known STS markers.
77
DIAGNOSTIC APPLICATIONS OF DIAGNOSTIC APPLICATIONS OF PCR CONTD.PCR CONTD.
Identifying genetic mutations.Identifying genetic mutations.
Single strand conformational Single strand conformational polymorphism.polymorphism.
WAVE DNA Fragment Analysis System by WAVE DNA Fragment Analysis System by Transgenomic.Transgenomic.
PCR genotyping using sequence tagged PCR genotyping using sequence tagged sites.sites.
88
LABORATORY APPLICATIONS LABORATORY APPLICATIONS OF PCROF PCR
Subcloning DNA targets using PCR.Subcloning DNA targets using PCR. PCR mediated in vitro mutagenesis.PCR mediated in vitro mutagenesis. Amplification of differently expressed Amplification of differently expressed
gene sequences.gene sequences. Differential display reverse Differential display reverse
trascriptase PCRtrascriptase PCR
99
APPLICATIONS OF PCRAPPLICATIONS OF PCRThe PCR has widespread applications in microbiology.The PCR has widespread applications in microbiology.
General General ApplicationsApplications
Genetic Testing for Genetic Testing for analysis of genetic analysis of genetic mutations.mutations.
Tissue typing vital Tissue typing vital to organ transplantto organ transplant
Forensic Forensic ApplicationsApplications
Applications in Applications in MicrobiologyMicrobiology
Characterization of Characterization of HIV virusHIV virus
Early detection of Early detection of M.tuberculosisM.tuberculosis
Early detection of Early detection of drug resistance of drug resistance of M.tuberculosisM.tuberculosis
1010
APPLICATIONS OF PCRAPPLICATIONS OF PCR
Detection of HIV-1 & HIV-2 Detection of HIV-1 & HIV-2 Detection of the viral loadDetection of the viral load Detection of M.tuberculosis through Detection of M.tuberculosis through
amplicor,EMTD.amplicor,EMTD. Identification of antibiotic resistance Identification of antibiotic resistance
associated mutations in MDRTBassociated mutations in MDRTB Identification of Cytomegalovirus Identification of Cytomegalovirus Identification of enteroviruses in CSFIdentification of enteroviruses in CSF
1111
PCR and bacteria
PCR for the detection and identification of bacteria isolated from environmental samples, has been used with two ways:1.Fast detection and identification of bacterial strains isolated(by cell culture) from the environment (e.g. Differentiation of strains isolated from the environment in pathogenic and non pathogenic)2.Direct detection of pathogenic bacteria in environmental samples without previous cell culture
1212
Advantages of PCR against cell culture techniques in virus
detection Increased sensitivity in the detection of viruses. 50% improvement in sensitivity Large variety of viruses detected Short time of analysis compared to virus culture techniques Low cost concerning the cultures
1313
APPLICATIONS OF PCRAPPLICATIONS OF PCR
Detection of Vancomycin resistant Detection of Vancomycin resistant enterococci from perianal swabsenterococci from perianal swabs
Detection of Treponema pallidum Detection of Treponema pallidum DNADNA
Detection of Almost all parasites Detection of Almost all parasites Diagnosis of Varicella zoster virus Diagnosis of Varicella zoster virus
infectioninfection Application of PCR in vaccine product Application of PCR in vaccine product
development.development.
1414
APPLICATIONS OF PCRAPPLICATIONS OF PCR
Diagnosis of Mycoplasma Diagnosis of Mycoplasma pneumoniaepneumoniae
Diagnosis of Bordetella pertusis & Diagnosis of Bordetella pertusis & parapertusisparapertusis
Amplification of rRNA & detection of Amplification of rRNA & detection of PsittacosisPsittacosis
Detection of Legionella DNADetection of Legionella DNA Detection of BartonellaDetection of Bartonella Diagnosis of Lyme diseaseDiagnosis of Lyme disease
1515
APPLICATIONS OF PCRAPPLICATIONS OF PCR
Specific & sensitive mean to directly Specific & sensitive mean to directly detect Brucella specimendetect Brucella specimen
Diagnosis of Chlamydia pneumoniaeDiagnosis of Chlamydia pneumoniae PCR has a reliable & sensitive role in PCR has a reliable & sensitive role in
characterization of strains involved in characterization of strains involved in nosocomial infection outbreaks.nosocomial infection outbreaks.
Diagnosis of Tuleremia.Diagnosis of Tuleremia.
1616
Applications of PCRApplications of PCR
Neisseria gonorrhea and Chlamydia Neisseria gonorrhea and Chlamydia trachomatis are two of the most trachomatis are two of the most common sexually transmitted common sexually transmitted diseases. The infections are diseases. The infections are asymptomatic and can lead to pelvic asymptomatic and can lead to pelvic inflammatory disease, salpingitis in inflammatory disease, salpingitis in women, epididymitis in men, women, epididymitis in men, infertility, and ectopic pregnancy.infertility, and ectopic pregnancy.
1717
Applications of PCRApplications of PCR
Specimens include endocervical Specimens include endocervical swabs,urethral swabs, and urine swabs,urethral swabs, and urine samples.samples.
The swabs are placed in a vial with The swabs are placed in a vial with transport buffer containing transport buffer containing 50mM 50mM MgCL2 and sodium azide as a MgCL2 and sodium azide as a preservative.preservative.
1818
Applications of PCRApplications of PCR
The swab specimens can be stored 2-The swab specimens can be stored 2-30°C for 4 days or frozen at -20°C.30°C for 4 days or frozen at -20°C.
The urine samples are refrigerated at The urine samples are refrigerated at 2-8°C or stored at -20°C.2-8°C or stored at -20°C.
A target sequence is chosen for both, A target sequence is chosen for both, amplified with polymerase, and then amplified with polymerase, and then evaluated with an enzyme evaluated with an enzyme immunoassay.immunoassay.
1919
Applications of PCRApplications of PCR
The HIV-1 test is used as a monitor of The HIV-1 test is used as a monitor of the severity of the virus. The HIV-1 the severity of the virus. The HIV-1 causes a depletion of CD4+ T causes a depletion of CD4+ T lymphocytes, causing lymphocytes, causing immunodeficiency, multiple immunodeficiency, multiple opportunistic infections, opportunistic infections, malignancies, and death.malignancies, and death.
2020
Applications of PCRApplications of PCR
The HIV-1 specimen is plasma The HIV-1 specimen is plasma collected in EDTA that must be collected in EDTA that must be separated from the cells within 6 separated from the cells within 6 hours.hours.
Heparin cannot be used as an Heparin cannot be used as an anticoagulant because it inhibits anticoagulant because it inhibits PCR.PCR.
2121
Applications of PCRApplications of PCR
A 142 base target sequence in the HIV-A 142 base target sequence in the HIV-1 gag gene is converted from RNA to 1 gag gene is converted from RNA to complementary DNA, and to double complementary DNA, and to double stranded DNA using Thermus stranded DNA using Thermus thermophilus DNA polymerase in the thermophilus DNA polymerase in the presence of manganese and buffers, presence of manganese and buffers, which performs the reverse which performs the reverse transcription and the amplification transcription and the amplification steps simultaneously. steps simultaneously.
2222
Application of PCRApplication of PCR
Treatment for patients with Factor V Treatment for patients with Factor V Leiden mutations are to give lifelong Leiden mutations are to give lifelong coumadin.coumadin.
Women with the mutation should not Women with the mutation should not take oral contraceptives, and they take oral contraceptives, and they have increased risk of thrombosis have increased risk of thrombosis during pregnancy.during pregnancy.
2323
Application of PCRApplication of PCR
And Many And Many More More ApplicationsApplications
2424
Real-Time Real-Time PCRPCR
2525
Real-time PrinciplesReal-time Principles
* based on the detection and quantitation of a fluorescent reporter
* the first significant increase in the
amount of PCR product (CT - threshold
cycle) correlates to the initial amount of target template
2626
Real-Time PrinciplesReal-Time Principles
Three general methods for the quantitative assays:
1. Hydrolysis probes
(TaqMan, Beacons, Scorpions)
2. Hybridization probes
(Light Cycler)
3. DNA-binding agents
(SYBR Green)
2727
Real-time PCR advantagesReal-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products (high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays(3 picogram = one genome equivalent)
* detection is capable down to a 2-fold change
* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR(except equipment cost)
2828
What is Wrong with What is Wrong with Agarose Gels?Agarose Gels?
* Poor precision
* Low sensitivity
* Short dynamic range < 2 logs
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
* Ethidium bromide staining is not very quantitative
2929
Principles of Real-Time Principles of Real-Time Quantitative PCR TechniquesQuantitative PCR Techniques
SYBR Green I technique: SYBR Green I fluorescence is enormously increased upon binding to double-stranded DNA. During the extension phase, more and more
SYBR Green I will bind to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle more
fluorescence signal will be detected. (a) Hydrolysis probe technique: The hydrolysis probe is conjugated with a
quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. However, upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This results in the separation of the reporter and quencher fluorochrome and consequently the fluorescence of the reporter fluorochrome becomes detectable. During each consecutive PCR cycle this fluorescence will further increase because of the progressive and exponential accumulation of free reporter fluorochromes.
(b) Hybridization probes technique: In this technique one probe is labelled with a donor fluorochrome at the 3’ end and a second –adjacent- probe is labelled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity (1–5 nucleotides apart), the emitted light of the donor fluorochrome will excite the acceptor fluorochrome (FRET). This results in the emission of fluorescence, which subsequently can be detected during the annealing phase and first part of the extension phase of the PCR reaction. After each subsequent PCR cycle more hybridization probes can anneal, resulting in higher fluorescence signals.
3030
Real-time PCR disadvantagesReal-time PCR disadvantages
* not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
* * *
* intra- and inter-assay variation
* RNA lability
* DNA contamination (in mRNA analysis)
3131
Real-Time PCR ApplicationsReal-Time PCR Applications - I - I
* quantitation of gene expression
* array verification
* quality control and assay validation
* biosafety and genetic stability testing
* drug therapy efficacy / drug monitoring
* viral quantitation
* pathogen detection
3232
Real-Time PCR ApplicationsReal-Time PCR Applications - II - II
* DNA damage (microsatellite instability) measurement
* radiation exposure assessment
* in vivo imaging of cellular processes
* mitochondrial DNA studies
* methylation detection
* detection of inactivation at X-chromosome
* linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target numbers in small
samples, which is adaptable to high throughput applications in clinical diagnostics, biodefense, forensics, and DNA
sequencing
3333
Real-Time PCR ApplicationsReal-Time PCR Applications - III - III
* Determination of identity at highly polymorphic HLA loci
* Monitoring post transplant solid organ graft outcome
* Monitoring chimerism after HSCT
* Monitoring minimal residual disease after HSCT
* Genotyping (allelic discrimination)
- Trisomies and single-gene copy numbers
- Microdeletion genotypes
- Haplotyping
- Quantitative microsatellite analysis
- Prenatal diagnosis from fetal cells in maternal blood
- Intraoperative cancer diagnostics
3434
POLYMERASE
CHAIN
REACTION
3535
Double strand cDNA
AAAAA
TTTTTRT
AAAAA
TTTTTRT
RTAAAAA
TTTTT
Oligo dT primer is bound to mRNA
Reverse transcriptase
(RT) copies first cDNA strand
Reverse transcriptase digests and
displaces mRNA and copies
second strand of cDNA
Conversion of mRNA to cDNA by Reverse Transcription
3636
A. Double strand DNA
B. Denature96º
50º
C. Anneal primers
50º
D. Polymerase binds
72ºTaq
Taq
3737
72ºTaq
Taq
E. Copy strands
1
2
3
4
F. Denature
96º
First round of cDNA
synthesis (4 strands)
Taq
Taq
3838
1
2
3
4
50ºG. Anneal primers
3939
1
2
3
4
Taq
Taq
Taq
Taq
72º
H. Polymerase binds
4040
1
2
3
4
Taq
Taq
Taq
Taq
I. Copy strands
72º
Second round of cDNA
synthesis (8 strands)
4141
1
2
3
4
J. Denature at 96ºAnneal primers at 50º
4242
1
2
3
4
72º
K. Bind polymerase (not shown) and copy strands
Third round of cDNA
synthesis (16
strands)
4343
1
23
4
L. Denature at 96ºAnneal primers at 50º
4444
1
23
4
M. Copy strands at 72º
Fourth round of cDNA
synthesis (32
strands)
72º
4545
1
2
3
4
cDNA strands (32) are
now shown as
lines
4747
The Taqman probe. The red circle represents the The Taqman probe. The red circle represents the quenching dye that disrupts the observable signal quenching dye that disrupts the observable signal
from the reporter dye (green circle) when it is within from the reporter dye (green circle) when it is within a short distance. a short distance.
4848
The TaqMan® probe binds to the target DNA, and the primer The TaqMan® probe binds to the target DNA, and the primer binds as well. Because the primer is bound,binds as well. Because the primer is bound, Taq Taq polymerase polymerase
can now create a complementary strandcan now create a complementary strand..
4949
The reporter dye is released from the extending double-The reporter dye is released from the extending double-stranded DNA created by the Taq polymerase. Away from the stranded DNA created by the Taq polymerase. Away from the quenching dye, the light emitted from the reporter dye in an quenching dye, the light emitted from the reporter dye in an
excited state can now be observedexcited state can now be observed
5050
A graph printout of actual data found using A graph printout of actual data found using
the TaqMan® probe.the TaqMan® probe.
5151
Another three step view of theAnother three step view of the TaqMan® probe working: before the TaqMan® probe working: before the probe is met with the Taq polymerase, energy is transferred from a probe is met with the Taq polymerase, energy is transferred from a
short-wavelength fluorophore (green) to a long-wavelength short-wavelength fluorophore (green) to a long-wavelength fluorophore (red). When the polymerase adds nucleotides to the fluorophore (red). When the polymerase adds nucleotides to the template strand, it releases the short-wavelength fluorophore, template strand, it releases the short-wavelength fluorophore,
making it detectable and the long-wavelength undetectablemaking it detectable and the long-wavelength undetectable
5252
Another view of TaqMan® in action. The release from the Another view of TaqMan® in action. The release from the Quencher dye (red Q) in step 2 eventually causes the Reporter Quencher dye (red Q) in step 2 eventually causes the Reporter
dye (blue R) to be seen in step 4.dye (blue R) to be seen in step 4.
5353
A real-time PCR machine used at Colorado State. A real-time PCR machine used at Colorado State. Courtesy Courtesy lamar.colostate.edulamar.colostate.edu..
5454
5555
5656
5757
5858
5959
6060
FRET = Förster/fluorescence resonance FRET = Förster/fluorescence resonance energy transferenergy transfer
6161
DNA Polymerase 5' DNA Polymerase 5' Exonuclease ActivityExonuclease Activity
6262
Fluoresces when boundto dsDNA
6363
SYBR Green(1) At the beginning of amplification, the reaction mixture contains the
denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is
subtracted during computer analysis. (2) After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. (3) During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in
real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.
6464
Molecular BeaconsMolecular Beacons
6565
ScorpionsScorpions
6666
NESTED PCRNESTED PCR
6767
Figure 1.Figure 1. Nested PCR strategy. Segment of DNA with dots representing Nested PCR strategy. Segment of DNA with dots representing nondiscript DNA sequence of unspecified length. The double lines represent a nondiscript DNA sequence of unspecified length. The double lines represent a
large distance between the portion of DNA illustrated in this figure. The portions large distance between the portion of DNA illustrated in this figure. The portions of DNA shown with four bases in a row represent PCR primer binding sites, of DNA shown with four bases in a row represent PCR primer binding sites,
though real primers would be longer. though real primers would be longer.
6868
Figure 2.Figure 2. The first pair of PCR primers (blue with The first pair of PCR primers (blue with arrows) bind to the outer pair of primer binding arrows) bind to the outer pair of primer binding
sites and amplify all the DNA in between these two sites and amplify all the DNA in between these two sites. sites.
6969
Figure 3.Figure 3. PCR product after the first round PCR product after the first round of amiplificaiton. Notice that the bases of amiplificaiton. Notice that the bases
outside the PCR primer pair are not present outside the PCR primer pair are not present in the product. in the product.
7070
Figure 4.Figure 4. Second pair of Second pair of nested primersnested primers (red with arrows) (red with arrows) bind to the first PCR product. The binding sites for the bind to the first PCR product. The binding sites for the
second pair of primers are a few bases "internal" to the first second pair of primers are a few bases "internal" to the first primer binding sites. primer binding sites.
7171
Figure 5.Figure 5. Final PCR product after second Final PCR product after second round of PCR. The length of the product is round of PCR. The length of the product is
defined by the location of the internal primer defined by the location of the internal primer binding sites. binding sites.
7272
Questions ?
7373