Ppt.pcr appli.

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


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


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


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


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


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


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


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


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..

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 ?

Ppt.pcr appli.

Health & Medicine

Transcript of Ppt.pcr appli.