polymerase chain reaction: diagnostic application roche by salwa hassan teama

Polymerase Chain Reaction: Diagnostic Application

Roche

BySalwa Hassan Teama

Polymerase Chain Reaction: Diagnostic Application

BySalwa Hassan Teama

M.D. N.C.I. Cairo University, Egypt

ContentsContents Standard Polymerase Chain Reaction (PCR)Standard Polymerase Chain Reaction (PCR) Requirements of the reactionRequirements of the reaction Thermal Cycling Profile for Standard PCRThermal Cycling Profile for Standard PCR Number of CyclesNumber of Cycles PCR Phases: Three phases:PCR Phases: Three phases: PCR ProductsPCR Products PCR MethodsPCR Methods The Evolution of PCR to Real-TimeThe Evolution of PCR to Real-Time Polymerase Chain Reaction: UsesPolymerase Chain Reaction: Uses PCR protocols:PCR protocols: http://www.protocolonline.org/prot/Molecular_Biology/PCR/ http://www.protocolonline.org/prot/Molecular_Biology/PCR/

Molecular Biology Glossary onlineMolecular Biology Glossary online

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.hthttp://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.htmlml

Standard Polymerase Chain Standard Polymerase Chain Reaction (PCR)Reaction (PCR)

Polymerase chain reaction is a technique for in vitro Polymerase chain reaction is a technique for in vitro amplification of specific DNA sequences via the temperatureamplification of specific DNA sequences via the temperature mediated DNA polymerase enzyme by simultaneous primer mediated DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA.extension of complementary strands of DNA.

PCR is an simple methods for making multiple copies of a DNA PCR is an simple methods for making multiple copies of a DNA sequence. Developed by researchers at cetus Corporation sequence. Developed by researchers at cetus Corporation ((Saiki et al., 1985) ; (Mullis and Faloona. 1987).Saiki et al., 1985) ; (Mullis and Faloona. 1987). PCR uses a PCR uses a thermostable DNA polymerase to produce a 2 fold amplification thermostable DNA polymerase to produce a 2 fold amplification of target genetic material with each temperature cycle. The of target genetic material with each temperature cycle. The PCR uses two oligonucleotide primer that are complementary PCR uses two oligonucleotide primer that are complementary to nucleic acid sequences flanking the target areato nucleic acid sequences flanking the target area , it has , it has become the most widely used nucleic acid amplification become the most widely used nucleic acid amplification technology and gold standard for amplification processes in technology and gold standard for amplification processes in diagnosisdiagnosis..

CroptechnologyCroptechnology

The polymerase chain reaction is a test The polymerase chain reaction is a test tube system for DNA replication that tube system for DNA replication that allows a "target" DNA sequence to be allows a "target" DNA sequence to be selectively amplified, several million-selectively amplified, several million-fold in just a few hours. RNA can be fold in just a few hours. RNA can be amplified if converted to cDNA by amplified if converted to cDNA by reverse transcriptase.reverse transcriptase.

Starting materials for gene analysis may be:Starting materials for gene analysis may be:•Genomic DNAGenomic DNA•RNA RNA •Nucleic acid from archival materialNucleic acid from archival material•Cloned DNA Cloned DNA •PCR productsPCR products

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Requirements of the reactionRequirements of the reaction

Template DNA: previously isolated and purifiedTemplate DNA: previously isolated and purified

Two primers: to flank the target sequenceTwo primers: to flank the target sequence

Four normal deoxynucleosides (dNTPs) : to provide energy Four normal deoxynucleosides (dNTPs) : to provide energy and nucleosides for the synthesis of DNAand nucleosides for the synthesis of DNA

Buffer system containing magnesiumBuffer system containing magnesium

DNA polymerase ( thermostable or heat-stable Taq polymerase DNA polymerase ( thermostable or heat-stable Taq polymerase isolated and purified from Thermus aquaticus, a bacterium lives isolated and purified from Thermus aquaticus, a bacterium lives in hot springs) in hot springs)

Template DNA :Template DNA : Sample preparation by DNA extraction. The Sample preparation by DNA extraction. The quality of the template influences the outcome of the PCR. If large quality of the template influences the outcome of the PCR. If large amount of RNA in DNA template can chelate Mg+ and reduce the amount of RNA in DNA template can chelate Mg+ and reduce the yield of the PCR. Also impure templates may contain polymerase yield of the PCR. Also impure templates may contain polymerase inhibitors that decrease the efficiency of the reaction. The integrity inhibitors that decrease the efficiency of the reaction. The integrity of the template is also important. Template DNA should be of high of the template is also important. Template DNA should be of high molecular weight. To check the size and quality, run an aliquot on molecular weight. To check the size and quality, run an aliquot on an agarose gel.an agarose gel.

The amount of template in a reaction strongly influences The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for performance in PCR. The recommended amount of template for standard PCR is:standard PCR is:

The maximum amount of :The maximum amount of : Human genomic DNA should be up to 500 ngHuman genomic DNA should be up to 500 ng 1-10 ng bacterial DNA1-10 ng bacterial DNA 0.1-1 ng plasmid DNA0.1-1 ng plasmid DNA



Primers:Primers: Oligonucleotide primers are synthesized by Oligonucleotide primers are synthesized by the DNA synthesizers. They are generally synthesized the DNA synthesizers. They are generally synthesized in the range 18-30 nucleotides. Typical primers are 18-in the range 18-30 nucleotides. Typical primers are 18-28 nucleotides in length having 50-60% GC 28 nucleotides in length having 50-60% GC composition. The calculated Tcomposition. The calculated Tmm for a given primer pair for a given primer pair should be balanced. Primer concentration between 0.1 should be balanced. Primer concentration between 0.1 and 0.6 and 0.6 m are generally optimal. Higher primer m are generally optimal. Higher primer concentration may promote mispriming and concentration may promote mispriming and accumulation of non specific product and may increase accumulation of non specific product and may increase the probability of generating a template independent the probability of generating a template independent artifact termed primer-dimer. Lower primer artifact termed primer-dimer. Lower primer concentration may be exhausted before the reaction is concentration may be exhausted before the reaction is completed resulting in lower yields of desired product.completed resulting in lower yields of desired product.


Buffer system:Buffer system: The standard PCR buffer contains The standard PCR buffer contains1.1. 1.5 mM MgCL21.5 mM MgCL22.2. 10 mM Tris HCl (PH 8.4)10 mM Tris HCl (PH 8.4)3.3. 50 mM50 mM KClKCl4.4. 100 100 g/ml gelatin or BSA (bovine serum albumin) g/ml gelatin or BSA (bovine serum albumin)

Mg concentration affects the reaction such that too Mg concentration affects the reaction such that too

little reduces yield and too much increases non little reduces yield and too much increases non specific amplification. The optimal MgClspecific amplification. The optimal MgCl22 concentration may vary from approximately 1mM-concentration may vary from approximately 1mM-5mM, 1.5 mM is optimal in most cases.5mM, 1.5 mM is optimal in most cases.

Requirements of the reactionRequirements of the reaction dNTP The final concentration of dNTPs should be 50-500 dNTP The final concentration of dNTPs should be 50-500 M M

(each dNTP).(each dNTP). They are usually included at conc. of 200 They are usually included at conc. of 200 M for each M for each

nucleotide. Higher concentration promote misincorporation by nucleotide. Higher concentration promote misincorporation by polymerase.polymerase. Always use balanced solution of all four dNTPs to Always use balanced solution of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP mixtures will minimize polymerase error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity. For carry over prevention reduce Taq DNA Polymerase fidelity. For carry over prevention a higher concentration of dUTP is usually used in place of dTTP.a higher concentration of dUTP is usually used in place of dTTP.

N.B.N.B. If you increase the concentration of dNTP you must increase If you increase the concentration of dNTP you must increase

Mg+ concentration. Increased in dNTPMg+ concentration. Increased in dNTP concentration reduce concentration reduce free Mg+, thus interfering with polymerase activity and decrease free Mg+, thus interfering with polymerase activity and decrease primer annealing.primer annealing.


Taq PolymeraseTaq Polymerase The most widely characterized polymerase is that from Thermus The most widely characterized polymerase is that from Thermus

aquaticus (Taq), which is a thermophilic bacterium lives in hot aquaticus (Taq), which is a thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C springs and capable of growing at 70 -75 C . The purified . The purified protein (Taq enzyme) has a molecular weight of 94 Kd, and has protein (Taq enzyme) has a molecular weight of 94 Kd, and has an optimum polymerization temperature of 70 – 80 C an optimum polymerization temperature of 70 – 80 C . The . The enzyme loses its activity, but is not denatured, at temperature enzyme loses its activity, but is not denatured, at temperature above 90 C above 90 C , and its activity is maintained on return to lower , and its activity is maintained on return to lower temprature. temprature.

0.5 – 2 units/50 0.5 – 2 units/50 l l reaction. Too little will limit the amount of reaction. Too little will limit the amount of product, while too much can produce unwanted non specific product, while too much can produce unwanted non specific products.products.

Thermal Cycling Profile for Thermal Cycling Profile for Standard PCRStandard PCR

Initial DenaturationInitial Denaturation

Initial heating of the PCR mixture for 2 minutes at 94- Initial heating of the PCR mixture for 2 minutes at 94- 95C 95C is enough to completely denature complex is enough to completely denature complex genomic DNA so that the primer can anneal to the genomic DNA so that the primer can anneal to the template as the reaction mix is cooled. If the template template as the reaction mix is cooled. If the template DNA is only partially denatured, it will tend to snap-DNA is only partially denatured, it will tend to snap-back very quickly, preventing efficient primer back very quickly, preventing efficient primer annealing and extension or leading to self priming annealing and extension or leading to self priming which can lead to false positive result. which can lead to false positive result.

Each cycle includes three successive steps:Each cycle includes three successive steps: Denaturation:Denaturation: One to several minutes at 94-96 COne to several minutes at 94-96 C, ,

during which the DNA is denatured into single strands.during which the DNA is denatured into single strands. Annealing:Annealing: One to several minutes at 50-65 C One to several minutes at 50-65 C , during , during

which the primers hybridize or "anneal" (by way of which the primers hybridize or "anneal" (by way of hydrogen bonds) to their complementary sequences on hydrogen bonds) to their complementary sequences on either side of the target sequence; andeither side of the target sequence; and

Extention:Extention: One to several minutes at 72 C One to several minutes at 72 C , during , during

which the polymerase binds and extends a which the polymerase binds and extends a complementary DNA strand from each primer. complementary DNA strand from each primer.

Thermal Cycling Profile for Standard PCRThermal Cycling Profile for Standard PCR

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During PCR, high temperature is used to separate the DNA During PCR, high temperature is used to separate the DNA molecules into single strands, and synthetic sequences of single-molecules into single strands, and synthetic sequences of single-stranded DNA (20-30 nucleotides) serve as primers. stranded DNA (20-30 nucleotides) serve as primers. Two different primer sequences are used to bracket the target Two different primer sequences are used to bracket the target region to be amplified. One primer is complementary to one DNA region to be amplified. One primer is complementary to one DNA strand at the beginning of the target region; a second primer is strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the complementary to a sequence on the opposite DNA strand at the end of the target region.end of the target region.The primer are arranged so that each primer extension reaction The primer are arranged so that each primer extension reaction directs the synthesis of DNA towards the other. directs the synthesis of DNA towards the other.

Primer AnnaPrimer annealing

Roche Molecular Biochemicals: PCR Application ManualRoche Molecular Biochemicals: PCR Application Manual

As amplification proceeds , the DNA sequence As amplification proceeds , the DNA sequence between primers doubles after each cycles (The between primers doubles after each cycles (The amplification of the target sequence proceeding in an amplification of the target sequence proceeding in an exponential fashion (1 2 4 8 exponential fashion (1 2 4 8 16…………….)16…………….)


Number of CyclesNumber of Cycles The number of cycles required for optimum amplification varies The number of cycles required for optimum amplification varies

depending on the amount of the starting material. In optimal depending on the amount of the starting material. In optimal reaction, less than 10 template molecules can be amplified in reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. Most PCR should , gel stained with ethidium bromide. Most PCR should , Therefore, include only 25 – 35 cycles. As cycle increases, Therefore, include only 25 – 35 cycles. As cycle increases, nonspecific products can accumulate. After 20- 40 cycles of nonspecific products can accumulate. After 20- 40 cycles of heating and cooling build up over a million copies of original heating and cooling build up over a million copies of original DNA molecules.DNA molecules.

Post extension and holdingPost extension and holding Cycling should conclude with a final extension at 72 c Cycling should conclude with a final extension at 72 c for 5 for 5

minute to promote completion of partial extension products and minute to promote completion of partial extension products and then holding at 4 c then holding at 4 c ..

Thermal Cycling Profile for Thermal Cycling Profile for Standard PCRStandard PCR

94 C Den.

Ann.

Ext. Post- Ext.

Holding

Hot start timeOne cycle repeated 25-35 timesOne cycle repeated 25-35 times Post-extension time

72C

54 C

4 C

Exponential:Exponential: Exact doubling of product is accumulating Exact doubling of product is accumulating

at every cycle (assuming 100% reaction efficiency). The at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.reaction is very specific and precise.

Linear :Linear : The reaction components are being consumed, The reaction components are being consumed, the reaction is slowing, and products are starting to the reaction is slowing, and products are starting to degrade.degrade.

Plateau:Plateau: (End-Point: Gel detection for traditional methods): (End-Point: Gel detection for traditional methods): The reaction has stopped, no more products are being The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin made and if left long enough, the PCR products will begin to degrade.to degrade.

PCR Phases: three phases:

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PCR Phases: Three Phases

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Plateau EffectPlateau Effect Plateau effectPlateau effect is used to describe the attenuation in the is used to describe the attenuation in the

exponential rate of product accumulation that occurs during exponential rate of product accumulation that occurs during the late PCR cycles. Depending on reaction conditions and the late PCR cycles. Depending on reaction conditions and thermal cycling one or more of the following may influence thermal cycling one or more of the following may influence plateau:plateau:

1.1. Utilization of substrates (dNTP or primers)Utilization of substrates (dNTP or primers)2.2. Stability of reactants (dNTP or enzyme)Stability of reactants (dNTP or enzyme)3.3. End product inhibitionEnd product inhibition4.4. Competition of reactants by non specific products or primer –Competition of reactants by non specific products or primer –

dimerdimer5.5. Incomplete denaturation/ strand separation of product at high Incomplete denaturation/ strand separation of product at high

product concentrationproduct concentration

PCR ProductsPCR Products

Following amplification, the Following amplification, the PCRPCR products are usually loaded into wells of products are usually loaded into wells of an agarose gel and electrophoresedan agarose gel and electrophoresed . .

Gel electrophoresis is a method used to separate or purify samples of Gel electrophoresis is a method used to separate or purify samples of DNA , RNA , or protein. A gel is made by dissolving agrose in buffer DNA , RNA , or protein. A gel is made by dissolving agrose in buffer solution, which is then allowed to set in a gel tray. The gel tray has solution, which is then allowed to set in a gel tray. The gel tray has combs attached to create wells in the gel, the samples are prepared combs attached to create wells in the gel, the samples are prepared and added to the well, and then an electric current is run through the gel and added to the well, and then an electric current is run through the gel apparatus. The DNA fragments are separated by charge (e.g. large apparatus. The DNA fragments are separated by charge (e.g. large fragment move more slowly than small fragments) and the relative sizes fragment move more slowly than small fragments) and the relative sizes of fragments are determined by comparing to a standard DNA ladderof fragments are determined by comparing to a standard DNA ladder..

Since Since PCRPCR amplifications can generate microgram quantities of product, amplifications can generate microgram quantities of product, amplified fragments can be visualized easily following staining with a amplified fragments can be visualized easily following staining with a chemical stain such as chemical stain such as ethidim bromideethidim bromide..

DNA ladder WellGel Electrophoresis

PCR MethodsPCR Methods

Reverse transcriptase-PCR (RT-PCR): Reverse transcriptase-PCR (RT-PCR):

PCR may be performed with RNA as a starting material. PCR may be performed with RNA as a starting material. RT-PCR, one of the most sensitive methods for the detection RT-PCR, one of the most sensitive methods for the detection

and analysis of rare mRNA transcripts or other RNA present in and analysis of rare mRNA transcripts or other RNA present in low abundance. low abundance.

RNA cannot serve as a template for PCR, so it must be first RNA cannot serve as a template for PCR, so it must be first

transcribed into cDNA with reverse transcriptase from Moloney transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified.cDNA copy is then amplified.

The technique is usually initiated by mixing short (12-18 The technique is usually initiated by mixing short (12-18 base) polymers of thymidine (oligo dT) with messenger base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand oligo dT as a primer to synthesize so-called first-strand cDNAcDNA . .

Reverse transcription polymerase chain reaction is widely Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases and, used in the diagnosis of genetic diseases and, quantitativelyquantitatively, in the determination of the abundance , in the determination of the abundance of specific different RNA molecules within a cell or of specific different RNA molecules within a cell or tissuetissue..

Reverse transcriptase-PCR (RT-PCR):


Reverse transcriptase-PCR (RT-PCR): Roche Molecular Biochemicals: PCR Roche Molecular Biochemicals: PCR

Application ManualApplication Manual

PCR MethodsPCR Methods

Nested-PCRNested-PCR is used to increase the specificity of the is used to increase the specificity of the PCR technique; two rounds of PCR are performed PCR technique; two rounds of PCR are performed consecutively, using two different pairs of primers. The consecutively, using two different pairs of primers. The known sequence is used to design two pairs of known sequence is used to design two pairs of primers. The second round primers primers. The second round primers (internal)(internal) are are located within the desired amplification product located within the desired amplification product produced by the first round primers produced by the first round primers (external).(external). It is It is highly unlikely that any region of DNA other than the highly unlikely that any region of DNA other than the intended target will allow sequential amplification with intended target will allow sequential amplification with both sets of primers. both sets of primers.

Quantitative PCRQuantitative PCR:: The determination or quantitation of the number of The determination or quantitation of the number of

copies of a given gene achieves accurate copies of a given gene achieves accurate estimation of DNA and RNA targets.estimation of DNA and RNA targets.

Hot-start PCRHot-start PCR - - to reduce non-specific to reduce non-specific amplification.amplification.

Multiplex-PCR Multiplex-PCR Mutagenesis by PCR.Mutagenesis by PCR. Inverse PCR Inverse PCR Asymmetric PCRAsymmetric PCR.. In Situ PCRIn Situ PCR..

PCR Methods

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Advantages of PCRAdvantages of PCR:: Useful non- invasive procedureUseful non- invasive procedure . .

Simplicity of the procedure.Simplicity of the procedure. Sensitivity of the PCR.Sensitivity of the PCR.

Disadvantages of PCR:Disadvantages of PCR: False positive results (cross contamination).False positive results (cross contamination). False negative results (rare of circulating fetal False negative results (rare of circulating fetal

cells).cells).

Traditional PCR has advanced from detection at the end-point of Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-the reaction to detection while the reaction is occurring (Real-Time).Time).

The real time system reduces the time required for PCR The real time system reduces the time required for PCR amplification and analysis from hours to minutes, it is perfectly amplification and analysis from hours to minutes, it is perfectly suited to:suited to:

Monitor amplification online and in real-timeMonitor amplification online and in real-time Quickly and accurately quantify results Quickly and accurately quantify results Analyze melting characteristics of PCR productAnalyze melting characteristics of PCR product RealReal--time PCR uses a fluorescent reporter signal to measure time PCR uses a fluorescent reporter signal to measure

the amount of amplicon as it is generatedthe amount of amplicon as it is generated.. This kinetic PCR This kinetic PCR allows for data collection after each cycle of PCR instead of allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cyclesonly at the end of the 20 to 40 cycles..

The Evolution of PCR to Real-Time

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The Evolution of The Evolution of PCR to Real-The Evolution of PCR to Real-TimeTime

End point detection

The recent development of The recent development of real time PCR clearly demonstrates real time PCR clearly demonstrates many advantages over other existing many advantages over other existing

method with: method with: high accuracy high accuracy

wide dynamic range wide dynamic range specificity specificity sensitivity sensitivity

reduced carry over contamination and rapid reduced carry over contamination and rapid accurate and simultaneous quantification of accurate and simultaneous quantification of

multiple samplesmultiple samples..

The Evolution of PCR to Real-Time

Polymerase Chain Reaction clearly has Polymerase Chain Reaction clearly has the potential to become the routine the potential to become the routine laboratory method for diagnosis of a laboratory method for diagnosis of a

variety of human disordersvariety of human disorders..

Detection of malignant diseases by Detection of malignant diseases by PCRPCR

The detection of leukemia and lymphomas by The detection of leukemia and lymphomas by the PCR method is currently the highest the PCR method is currently the highest developed in cancer research and is already developed in cancer research and is already being used routinely .being used routinely .

PCR assays can be performed directly on genomic PCR assays can be performed directly on genomic

DNA samples to detect translocation-specific DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least malignant cells at a sensitivity which is at least

10,000 fold higher than other methods10,000 fold higher than other methods. .

t(8;21) translocation or AML1-ETO fusion genet(8;21) translocation or AML1-ETO fusion gene t(15;17) translocation or PML-RARA fusion genet(15;17) translocation or PML-RARA fusion gene INV(16) or MYH11-CBFB fusion geneINV(16) or MYH11-CBFB fusion gene t(9;22) translocation or BCR-ABL fusion gene (p210 and p185t(9;22) translocation or BCR-ABL fusion gene (p210 and p185 FLT3 MutationsFLT3 Mutations BCR-ABL MutationsBCR-ABL Mutations

Polymerase Chain Reaction: Uses

Polymerase Chain Reaction: UsesPolymerase Chain Reaction: Uses

Recurrence of hematological Recurrence of hematological cancers has also been evaluatedcancers has also been evaluated- To measure the risk of relapse of T lineage acute To measure the risk of relapse of T lineage acute

lymphoblastic leukemia in children, detection and lymphoblastic leukemia in children, detection and quantitation of residual leukemic cells that harbor the quantitation of residual leukemic cells that harbor the TAL deletion.TAL deletion.

Monitoring the MRD in leukemia and lymphoma patients Monitoring the MRD in leukemia and lymphoma patients by assessing PRAME (Preferentially expressed antigen by assessing PRAME (Preferentially expressed antigen of melanoma) in peripheral blood samples.of melanoma) in peripheral blood samples.

One area where the PCR technique will undoubtedly One area where the PCR technique will undoubtedly become a routine method, is the become a routine method, is the detection of detection of infectious agentsinfectious agents, such as , such as pathogenic bacteria, pathogenic bacteria, viruses or protozoa. PCR provides a considerable viruses or protozoa. PCR provides a considerable advantageadvantage over other commonly used methods. This over other commonly used methods. This is especially true for the identification of non-is especially true for the identification of non-cultivatable or slow-growing microorganisms such as cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria etc. or viruses, mycobacteria, anaerobic bacteria etc. or viruses, where tissue culture assays and animal models have where tissue culture assays and animal models have to be used or which cannot be cultivated at all. to be used or which cannot be cultivated at all.

Polymerase Chain Reaction: Uses

Polymerase Chain Reaction: UsesPolymerase Chain Reaction: Uses

The basis for PCR diagnostic applications in The basis for PCR diagnostic applications in microbiologymicrobiology is the is the detection of infectious agents and the discrimination of non-detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains (e.g. E.coli) by virtue of pathogenic from pathogenic strains (e.g. E.coli) by virtue of specific genes. specific genes.

PCR primers have also been reported for intracellular parasites PCR primers have also been reported for intracellular parasites like T.gondij , P.falciparum and for different strains of like T.gondij , P.falciparum and for different strains of Trypanosoma,….Trypanosoma,….

In virology a large number of PCR assays have been described In virology a large number of PCR assays have been described for the Human immunodeficiency viruses , CMV , HBV ,HSV and for the Human immunodeficiency viruses , CMV , HBV ,HSV and others………..others………..

Polymerase Chain Reaction: UsesPolymerase Chain Reaction: Uses**Major role in the human genome project.Major role in the human genome project.

** Single point mutations can be detected by modified PCR techniques Single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-single-strand such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis. conformational polymorphisms (PCR-SSCP) analysis.

**Detection of variation and mutation in genes using primers containing Detection of variation and mutation in genes using primers containing sequences that were not completely complementary to the template.sequences that were not completely complementary to the template.

** Identify the level of expression of genes in extremely small samples Identify the level of expression of genes in extremely small samples of material, e.g. tissues or cells from the body by reverse of material, e.g. tissues or cells from the body by reverse transcription-PCR (RT-PCR).transcription-PCR (RT-PCR).

**Amplification of archival and forensic materialAmplification of archival and forensic material

Polymerase Chain Reaction: Polymerase Chain Reaction: UsesUses

** Extending PCR to the amplification of more than one sequence Extending PCR to the amplification of more than one sequence at a time ( multiplex PCR) made it possible to compare two or at a time ( multiplex PCR) made it possible to compare two or more complex genomes, for instance to detect chromosomal more complex genomes, for instance to detect chromosomal imbalances.imbalances.

** Combining in situ hybridization with PCR made possible the Combining in situ hybridization with PCR made possible the localization of single nucleic acid sequences on one localization of single nucleic acid sequences on one chromosome within an eukaryotic organism.chromosome within an eukaryotic organism.

** Detection of micro-metastasis in blood, lymph nodes and bone Detection of micro-metastasis in blood, lymph nodes and bone marrow.marrow.

** HLA Typing. HLA Typing. * * Analyzing the expression of cytokeratin-18 mRNA in Analyzing the expression of cytokeratin-18 mRNA in

gastrointestinal carcinoma cell lines. gastrointestinal carcinoma cell lines. ** DNA analysis for genetic disease diagnosis. DNA analysis for genetic disease diagnosis.

Application of real time PCR in Application of real time PCR in molecular diagnosismolecular diagnosis

Clinical MicrobiologyClinical Microbiology Viral load (HIV,HCV,HBV,…)Viral load (HIV,HCV,HBV,…) Bacterial load (Salmonella, Mycobacterium,..)Bacterial load (Salmonella, Mycobacterium,..) Fungal load( Candida, Cryptococcus, Aspergillus,….)Fungal load( Candida, Cryptococcus, Aspergillus,….)Food microbiologyFood microbiology Bacterial load (Listeria, Salmonella, Campylobacter,…)Bacterial load (Listeria, Salmonella, Campylobacter,…)Clinical OncologyClinical Oncology Minimal residual diseaseMinimal residual disease Chromosomal translocationsChromosomal translocations Single nucleotide polymorphism (SNPs)Single nucleotide polymorphism (SNPs)Gene therapyGene therapy Gene transfer estimationGene transfer estimation Biodistribution of vectorBiodistribution of vector

Gene expressionGene expression Cytokines, receptors,……..Cytokines, receptors,……..

ConclusionConclusionPolymerase Chain Reaction clearly has the potential to Polymerase Chain Reaction clearly has the potential to

become the routine laboratory method for diagnosis of become the routine laboratory method for diagnosis of a variety of human disorders. Most clearly, the a variety of human disorders. Most clearly, the detection of infectious agents surpasses current detection of infectious agents surpasses current routine methods.routine methods.

PCR has very quickly become an essential tool for PCR has very quickly become an essential tool for improving human health and human lifeimproving human health and human life..

Velasco JVelasco J. . A new view of malignancyA new view of malignancy New York TimesApril 9, 2002New York TimesApril 9, 2002.... Watson JD, Crick FHCWatson JD, Crick FHC. . Molecular structure of nucleic acidsMolecular structure of nucleic acids. . NatureNature. 1953;171:737–738. . 1953;171:737–738. PubMedPubMed Osler, WOsler, W. . The Principles and Practice of MedicineThe Principles and Practice of Medicine. . New YorkNew York: : Appleton; 1892Appleton; 1892. . Stites DP . Medical immunology. Section II. Page 270Stites DP . Medical immunology. Section II. Page 270 Amr Karim. Workshop on molecular biology and genetic engineering. Faculty of science . Ain Amr Karim. Workshop on molecular biology and genetic engineering. Faculty of science . Ain

Shams UniversityShams University WWW.medscape.comWWW.medscape.com www. pubmedcentral.nihwww. pubmedcentral.nih www.Rochewww.Roche Molecular Biochemicals: PCR Applications Manual Molecular Biochemicals: PCR Applications Manual www.Rochewww.Roche Molecular Biochemicals: PCR Techniques Molecular Biochemicals: PCR Techniques www. AppliedBiosystem.COM Real Time PCRwww. AppliedBiosystem.COM Real Time PCR Watson JD, Crick FHCWatson JD, Crick FHC. . Molecular structure of nucleic acidsMolecular structure of nucleic acids. . NatureNature. 1953;171:737–738. [. 1953;171:737–738. [PubMedPubMed]] Osler, WOsler, W. . The Principles and Practice of MedicineThe Principles and Practice of Medicine. . New YorkNew York: : Appleton; 1892Appleton; 1892 http://en.wikipedia.org/wiki/RT-PCRhttp://en.wikipedia.org/wiki/RT-PCR http://www.ma.uni-heidelberg.de/inst/ikc/molekularbiologie/rt-pcr.jpghttp://www.ma.uni-heidelberg.de/inst/ikc/molekularbiologie/rt-pcr.jpg

References & Online Further Reading

polymerase chain reaction: diagnostic application roche by salwa hassan teama

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