Polymerase chain reaction (PCR) <ul><li>A related, more recent Nobel Prize (Chemistry, 1993) was given for the Polymerase Chain Reaction (PCR) to Kary Mullis </li></ul><ul><li>This puts together the concepts of DNA synthesis catalyzed by a polymerase, denaturation, and annealing. </li></ul>"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."
PCR AGAROSE GEL ELECTROPHORESIS THE FINAL PRODUCT 3 TO 4 HOURS U V VISUALIZATION
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 expensive 4. A few bacteria in the environment are viable but not culturable.
DIAGNOSTIC APPLICATIONS OF PCR <ul><li>THERE ARE THREE PRIMARY DIAGNOSTIC APPLICATIONS OF PCR </li></ul><ul><li>Detecting pathogens using genome – specific primer pairs. </li></ul><ul><li>Screening specific genes for unknown mutations. </li></ul><ul><li>Genotyping using known STS markers. </li></ul>
DIAGNOSTIC APPLICATIONS OF PCR CONTD. <ul><li>Identifying genetic mutations. </li></ul><ul><li>Single strand conformational polymorphism. </li></ul><ul><li>WAVE DNA Fragment Analysis System by Transgenomic. </li></ul><ul><li>PCR genotyping using sequence tagged sites. </li></ul>
LABORATORY APPLICATIONS OF PCR <ul><li>Subcloning DNA targets using PCR. </li></ul><ul><li>PCR mediated in vitro mutagenesis. </li></ul><ul><li>Amplification of differently expressed gene sequences. </li></ul><ul><li>Differential display reverse trascriptase PCR </li></ul>
APPLICATIONS OF PCR The PCR has widespread applications in microbiology. <ul><li>General Applications </li></ul><ul><li>Genetic Testing for analysis of genetic mutations. </li></ul><ul><li>Tissue typing vital to organ transplant </li></ul><ul><li>Forensic Applications </li></ul><ul><li>Applications in Microbiology </li></ul><ul><li>Characterization of HIV virus </li></ul><ul><li>Early detection of M.tuberculosis </li></ul><ul><li>Early detection of drug resistance of M.tuberculosis </li></ul>
APPLICATIONS OF PCR <ul><li>Detection of HIV-1 & HIV-2 </li></ul><ul><li>Detection of the viral load </li></ul><ul><li>Detection of M.tuberculosis through amplicor,EMTD. </li></ul><ul><li>Identification of antibiotic resistance associated mutations in MDRTB </li></ul><ul><li>Identification of Cytomegalovirus </li></ul><ul><li>Identification of enteroviruses in CSF </li></ul>
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
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
APPLICATIONS OF PCR <ul><li>Detection of Vancomycin resistant enterococci from perianal swabs </li></ul><ul><li>Detection of Treponema pallidum DNA </li></ul><ul><li>Detection of Almost all parasites </li></ul><ul><li>Diagnosis of Varicella zoster virus infection </li></ul><ul><li>Application of PCR in vaccine product development. </li></ul>
APPLICATIONS OF PCR <ul><li>Diagnosis of Mycoplasma pneumoniae </li></ul><ul><li>Diagnosis of Bordetella pertusis & parapertusis </li></ul><ul><li>Amplification of rRNA & detection of Psittacosis </li></ul><ul><li>Detection of Legionella DNA </li></ul><ul><li>Detection of Bartonella </li></ul><ul><li>Diagnosis of Lyme disease </li></ul>
APPLICATIONS OF PCR <ul><li>Specific & sensitive mean to directly detect Brucella specimen </li></ul><ul><li>Diagnosis of Chlamydia pneumoniae </li></ul><ul><li>PCR has a reliable & sensitive role in characterization of strains involved in nosocomial infection outbreaks. </li></ul><ul><li>Diagnosis of Tuleremia. </li></ul>
Applications of PCR <ul><li>Neisseria gonorrhea and Chlamydia trachomatis are two of the most common sexually transmitted diseases. The infections are asymptomatic and can lead to pelvic inflammatory disease, salpingitis in women, epididymitis in men, infertility, and ectopic pregnancy. </li></ul>
Applications of PCR <ul><li>Specimens include endocervical swabs,urethral swabs, and urine samples. </li></ul><ul><li>The swabs are placed in a vial with transport buffer containing 50mM MgCL2 and sodium azide as a preservative. </li></ul>
Applications of PCR <ul><li>The swab specimens can be stored 2-30°C for 4 days or frozen at -20°C. </li></ul><ul><li>The urine samples are refrigerated at 2-8°C or stored at -20°C. </li></ul><ul><li>A target sequence is chosen for both, amplified with polymerase, and then evaluated with an enzyme immunoassay. </li></ul>
Applications of PCR <ul><li>The HIV-1 test is used as a monitor of the severity of the virus. The HIV-1 causes a depletion of CD4+ T lymphocytes, causing immunodeficiency, multiple opportunistic infections, malignancies, and death. </li></ul>
Applications of PCR <ul><li>The HIV-1 specimen is plasma collected in EDTA that must be separated from the cells within 6 hours. </li></ul><ul><li>Heparin cannot be used as an anticoagulant because it inhibits PCR. </li></ul>
Applications of PCR <ul><li>A 142 base target sequence in the HIV-1 gag gene is converted from RNA to complementary DNA, and to double stranded DNA using Thermus thermophilus DNA polymerase in the presence of manganese and buffers, which performs the reverse transcription and the amplification steps simultaneously. </li></ul>
Application of PCR <ul><li>Treatment for patients with Factor V Leiden mutations are to give lifelong coumadin. </li></ul><ul><li>Women with the mutation should not take oral contraceptives, and they have increased risk of thrombosis during pregnancy. </li></ul>
Application of PCR <ul><li>And Many More Applications </li></ul>
Real-time Principles * based on the detection and quantitation of a fluorescent reporter * the first significant increase in the amount of PCR product (C T - threshold cycle) correlates to the initial amount of target template
Real-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)
Real-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 10 10 -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)
What is Wrong with 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
<ul><li>Principles of Real-Time Quantitative PCR Techniques </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul>
Real-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)
Real-Time PCR Applications - 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
Real-Time PCR Applications - 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
Real-Time PCR Applications - 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 - Q uantitative microsatellite analysis - Prenatal diagnosis from fetal cells in maternal blood - Intraoperative cancer diagnostics
Double strand cDNA AAAAA TTTTT RT RT RT 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 AAAAA TTTTT AAAAA TTTTT
A. Double strand DNA 50 º Taq Taq B. Denature 96 º C. Anneal primers 50 º D. Polymerase binds 72 º
Taq Taq E. Copy strands First round of cDNA synthesis (4 strands) Taq Taq 72 º 1 2 3 4 F. Denature 96 º
1 2 3 4 Taq Taq Taq Taq I. Copy strands 72 º Second round of cDNA synthesis (8 strands)
1 2 3 4 J. Denature at 96 º Anneal primers at 50 º
1 2 3 4 72 º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands)
1 2 3 4 L. Denature at 96 º Anneal primers at 50 º
1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72 º
1 2 3 4 cDNA strands (32) are now shown as lines
1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size
The Taqman probe. The red circle represents the quenching dye that disrupts the observable signal from the reporter dye (green circle) when it is within a short distance.
The TaqMan® probe binds to the target DNA, and the primer binds as well. Because the primer is bound, Taq polymerase can now create a complementary strand .
The reporter dye is released from the extending double-stranded DNA created by the Taq polymerase. Away from the quenching dye, the light emitted from the reporter dye in an excited state can now be observed
A graph printout of actual data found using the TaqMan® probe.
Another three step view of the TaqMan® probe working: before the probe is met with the Taq polymerase, energy is transferred from a short-wavelength fluorophore (green) to a long-wavelength fluorophore (red). When the polymerase adds nucleotides to the template strand, it releases the short-wavelength fluorophore, making it detectable and the long-wavelength undetectable
Another view of TaqMan® in action. The release from the Quencher dye (red Q) in step 2 eventually causes the Reporter dye (blue R) to be seen in step 4.
A real-time PCR machine used at Colorado State. Courtesy lamar.colostate.edu .
FRET = Förster/fluorescence resonance energy transfer
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.
Figure 1. Nested PCR strategy. Segment of DNA with dots representing nondiscript DNA sequence of unspecified length. The double lines represent a 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, though real primers would be longer.
Figure 2. The first pair of PCR primers (blue with arrows) bind to the outer pair of primer binding sites and amplify all the DNA in between these two sites.
Figure 3. PCR product after the first round of amiplificaiton. Notice that the bases outside the PCR primer pair are not present in the product.
Figure 4. Second pair of nested primers (red with arrows) bind to the first PCR product. The binding sites for the second pair of primers are a few bases "internal" to the first primer binding sites.
Figure 5. Final PCR product after second round of PCR. The length of the product is defined by the location of the internal primer binding sites.