Polymerase chain reaction

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Contents
Polymerase Chain Reaction
PCR Reaction Components
Standard PCR Reaction
Avoiding Contamination
Thermal Cycling Profile for

Standard PCR
Gel Electrophoresis
PCR: Three phases
Variants of PCR
Polymerase Chain Reaction: Uses

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 Coping Machine for DNA Molecule
 Invented by Kary Mullis and

his colleagues in the 1983

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Polymerase Chain Reaction
PCR: Technique for in vitro (test tube) amplification of

specific DNA sequences via the temperature mediated. DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA.
PCR: This system for DNA replication that allows a "target" DNA sequence to be selectively amplified, several million-fold in just a few hours.

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PCR

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PCR reaction components
шаблон
A, G, C, T
Mg2+
(forward
and reverse)

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PCR reaction components
DNA template
Two primers
Four normal deoxynucleosides triphosphates
Buffer system
DNA polymerase I

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DNA Template
Integrity
High molecular weight
Purity
Pure
Amount
Human genomic DNA should be up to 500ng
Bacterial

DNA 1-10ng
Plasmid DNA 0.1-1ng

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Primers
Typical primers are 18-28 bases in length,
Having 40- 60% GC composition,
Have

a balanced distribution of G/C and A/T rich domains,
The calculated Tm for a given primer pair should be balanced (difference no more than 5 °C),
Primer concentration between 0.1 and 0.6 µM are generally optimal,
Contain no internal secondary structure,
Have a cytosine and guanine at the 3'-end because they form three hydrogen bonds with the matrix molecules, making a more stable hybridization

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Four Normal Deoxynucleosides Triphosphate
Final concentration of dNTPs should be 50-500 µM

(each dNTP). Usually included at conc. of 200 µM for each nucleotide.
Always use balanced solution of all four dNTPs to minimize polymerase error rate.

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Tris-HCl 10mM (10-50mM) for dissolution of nucleic acids
рH

8.3 (рH 8.3-8.8 at 20C°)
KCl 50mM promotes specificity of hybridization
MgCL2 1.5mM (0.5-10mM) for stabilizing of complex between primers and matrix and for increasing of exit the special product of PCR
Gelatin or Bovine Serum Albumin 100 µg/ml
frequent unfreezing-freezing at the temperature -20C

The standard PCR buffer contains:

Buffer System Containing Magnesium

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DNA Polymerase
The most widely characterized polymerase is that from Thermus aquaticus

(Taq), Thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C°,
Consist of a single polypeptide chain has a molecular weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C° (72 C°).
0.5 – 2 units/50µl reaction. Too little will limit the amount of products, while too much can produce unwanted non specific products.

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Enhance The Specificity and or Efficiency of a PCR
Betadine (antiseptic)
Bovine serum

albumin (for stabilizing of enzymes)
Dimethylysulfoxide for inhibition of connubium of initial
molecules of DNA
Glycerol
Pyrophosphate
Spermidine, Detergent, Gelatin,….

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Calculation of Melting Temperature
Tm= 2 C° X (number of A and

T bases)+4 C°X
(number of G and C bases).

Optimal annealing temperature are 5-10 C ° lower than Tm values of the primers .

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STANDARD PCR REACTION

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PCR

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AVOIDING CONTAMINATION

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Sample Handling
Use sterile techniques and always wear fresh gloves,
Always use new

or sterilized glassware, plasticware and pipettes to prepare the PCR reagents and template DNA,
Autoclave and sterilize all reagents and solution,
Have your own set of PCR reagent and Solution (store in small aliquots),
Positive and negative control should be included.

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Laboratory Facilities
Set up physically separated working places for:
Template preparation
Setting up PCR

reactions
Post PCR analysis
Use PCR only pipettes, micro-centrifuges and disposable gloves
Use aerosol resistant pipette tips
PCR reaction under a fume hood equipped with UV
LIGHT.

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Working with RNA
Do not touch a surface after putting the

gloves to avoid reintroduction of RNAse to decontaminated material.
Designate a special area for RNA work only.
Treat surface or benches and glassware with commercially available RNAse inactivating agents.

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Polymerase Chain Reaction

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Thermal Cycling Profile for Standard PCR
Initial Denaturation:
Initial heating of

the PCR mixture at 94- 95C within 2 min. is enough to completely denature complex genomic DNA.
Each cycle includes three successive steps: Denaturation, annealing and extension.
Post extension and holding:
Cycling should conclude with a final extension at 72 C° for 5 -15 minute to promote completion of partial extension products and then holding at 4 C°.

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Each cycle includes three successive steps:

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PCR

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Exponential Amplification
As amplification proceeds, the DNA sequence between primers doubles after

each cycle.
(The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16…………….) up to million of times the starting amount until enough is present to be seen by gel electrophoresis.

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

depending on the amount of the starting material.
Most PCR should, therefore, include only 25 – 35 cycles. As cycle increases, nonspecific products can accumulate.
After 20- 40 cycles of heating and cooling build up over a million copies of original DNA molecules.

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GEL ELECTROPHORESIS

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Agarose Gel Electrophoresis
It is a method used in biochemistry and molecular

biology to separate DNA, or RNA molecules based upon charge, size and shape.
Agarose is a polysaccharide derivative of agar.

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Gel Tray/ Loading

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PCR Product
DNA Molecular Marker
Amplified fragments can be visualized easily following

staining with a chemical stain such as ethidium bromide.
The DNA fragments are separated by charge and the relative sizes of fragments are determined by comparing to a standard DNA lad

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» Factors, affect the mobility of molecules in gel
Charge
Size
Shape
Buffer conditions
Gel concentration

and
Voltage

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PCR: Three Phases
Exponential: Exact doubling of product is accumulating at every

cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.
Linear: The reaction components are being consumed; the reaction is slowing, and products are starting to degrade.
Plateau: The reaction has stopped; no more products are being made and if left long enough; the PCR products will begin to degrade.

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PCR Phases

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Polymerase Chain Reaction
Advantages of PCR
Useful non- invasive procedure.
Simplicity of the procedure.
Sensitivity

of the PCR
Disadvantages of PCR
False positive results (cross contamination).
False negative results

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Variant PCR
Reverse transcriptase-PCR.
Nested-PCR.
Hot-start PCR.
Quantitative PCR.
Multiplex-PCR.
Mutagenesis by PCR.
Allele specific PCR.
…..

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Reverse Transcriptase - PCR
RT-PCR, one of the most sensitive methods for

the detection and analysis of rare mRNA transcripts or other RNA present in low abundance.
RNA cannot serve as a template for PCR.
RNA must be first transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified.

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RT- PCR

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Nested PCR
Nested PCR is a very specific PCR amplification.
Nested PCR use

two pairs (instead of one pair) of PCR primers are used to amplify a fragment.

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Nested - PCR

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Hot - Start PCR
Hot Start PCR significantly improves specificity, sensitivity and

yield of PCR.
The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems can be used.

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Hot - Start PCR

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Real Time PCR
Traditional PCR has advanced from detection at the end-point

of the reaction to detection while the reaction is occurring (Real-Time).
Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated . This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.

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Real Time PCR

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Infectious Diseases/ Cancer
Detection of infectious agents, such as Pathogenic bacteria, Viruses

or Protozoa.
Cancer
Detection of malignant diseases by PCR, Recurrence of hematological cancers has also been evaluated and
Detection of micro-metastasis in blood, lymph nodes and bone marrow.

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Genetic Desease
Single point mutations can be detected by modified PCR techniques

such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis.
Detection of variation and mutation in genes using primers containing sequences that were not completely complementary to the template.

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Prenatal Diagnosis
Prenatal sexing: Often required in families with inherited sex-linked diseases.
Prenatal

Diagnosis of diseases: Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA markers.

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Research
PCR is used in research laboratories in DNA cloning procedures, Southern

blotting, DNA sequencing, recombinant DNA technology.
Major role in the human genome project.

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Polymerase chain reaction

Содержание

POLYMERASE CHAIN REACTION

ContentsPolymerase Chain ReactionPCR Reaction ComponentsStandard PCR ReactionAvoiding ContaminationThermal Cycling Profile for

 Coping Machine for DNA Molecule Invented by Kary Mullis and

Polymerase Chain ReactionPCR: Technique for in vitro (test tube) amplification of

PCR

PCR reaction componentsшаблонA, G, C, TMg2+(forward and reverse)

PCR reaction componentsDNA templateTwo primersFour normal deoxynucleosides triphosphatesBuffer systemDNA polymerase I

DNA TemplateIntegrityHigh molecular weightPurityPureAmountHuman genomic DNA should be up to 500ngBacterial

PrimersTypical primers are 18-28 bases in length,Having 40- 60% GC composition,Have

Four Normal Deoxynucleosides TriphosphateFinal concentration of dNTPs should be 50-500 µM

Tris-HCl 10mM (10-50mM) for dissolution of nucleic acids рH

DNA PolymeraseThe most widely characterized polymerase is that from Thermus aquaticus

Enhance The Specificity and or Efficiency of a PCRBetadine (antiseptic)Bovine serum

Calculation of Melting TemperatureTm= 2 C° X (number of A and

STANDARD PCR REACTION

PCR

AVOIDING CONTAMINATION

Sample HandlingUse sterile techniques and always wear fresh gloves,Always use new

Laboratory FacilitiesSet up physically separated working places for:Template preparationSetting up PCR

Working with RNA Do not touch a surface after putting the

Polymerase Chain Reaction

Thermal Cycling Profile for Standard PCR Initial Denaturation: Initial heating of

Each cycle includes three successive steps:

PCR

Exponential AmplificationAs amplification proceeds, the DNA sequence between primers doubles after

Number of CyclesThe number of cycles required for optimum amplification varies

GEL ELECTROPHORESIS

Agarose Gel ElectrophoresisIt is a method used in biochemistry and molecular

Gel Tray/ Loading

PCR ProductDNA Molecular Marker Amplified fragments can be visualized easily following

» Factors, affect the mobility of molecules in gel ChargeSizeShapeBuffer conditionsGel concentration

PCR: Three PhasesExponential: Exact doubling of product is accumulating at every

PCR Phases

Polymerase Chain ReactionAdvantages of PCRUseful non- invasive procedure.Simplicity of the procedure.Sensitivity

Variant PCRReverse transcriptase-PCR.Nested-PCR.Hot-start PCR.Quantitative PCR.Multiplex-PCR.Mutagenesis by PCR.Allele specific PCR.…..

Reverse Transcriptase - PCRRT-PCR, one of the most sensitive methods for

RT- PCR

Nested PCRNested PCR is a very specific PCR amplification.Nested PCR use

Nested - PCR

Hot - Start PCRHot Start PCR significantly improves specificity, sensitivity and

Hot - Start PCR

Real Time PCRTraditional PCR has advanced from detection at the end-point

Real Time PCR

Infectious Diseases/ CancerDetection of infectious agents, such as Pathogenic bacteria, Viruses

Genetic DeseaseSingle point mutations can be detected by modified PCR techniques

Prenatal DiagnosisPrenatal sexing: Often required in families with inherited sex-linked diseases.Prenatal

ResearchPCR is used in research laboratories in DNA cloning procedures, Southern

Polymerase Chain Reaction

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Contents
Polymerase Chain Reaction
PCR Reaction Components
Standard PCR Reaction
Avoiding Contamination
Thermal Cycling Profile for

 Coping Machine for DNA Molecule
 Invented by Kary Mullis and

Polymerase Chain Reaction
PCR: Technique for in vitro (test tube) amplification of

PCR reaction components
шаблон
A, G, C, T
Mg2+
(forward
and reverse)

PCR reaction components
DNA template
Two primers
Four normal deoxynucleosides triphosphates
Buffer system
DNA polymerase I

DNA Template
Integrity
High molecular weight
Purity
Pure
Amount
Human genomic DNA should be up to 500ng
Bacterial

Primers
Typical primers are 18-28 bases in length,
Having 40- 60% GC composition,
Have

Four Normal Deoxynucleosides Triphosphate
Final concentration of dNTPs should be 50-500 µM

Tris-HCl 10mM (10-50mM) for dissolution of nucleic acids
рH

DNA Polymerase
The most widely characterized polymerase is that from Thermus aquaticus

Enhance The Specificity and or Efficiency of a PCR
Betadine (antiseptic)
Bovine serum

Calculation of Melting Temperature
Tm= 2 C° X (number of A and

Sample Handling
Use sterile techniques and always wear fresh gloves,
Always use new

Laboratory Facilities
Set up physically separated working places for:
Template preparation
Setting up PCR

Working with RNA
Do not touch a surface after putting the

Thermal Cycling Profile for Standard PCR
Initial Denaturation:
Initial heating of

Exponential Amplification
As amplification proceeds, the DNA sequence between primers doubles after

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

Agarose Gel Electrophoresis
It is a method used in biochemistry and molecular

PCR Product
DNA Molecular Marker
Amplified fragments can be visualized easily following

» Factors, affect the mobility of molecules in gel
Charge
Size
Shape
Buffer conditions
Gel concentration

PCR: Three Phases
Exponential: Exact doubling of product is accumulating at every

Polymerase Chain Reaction
Advantages of PCR
Useful non- invasive procedure.
Simplicity of the procedure.
Sensitivity

Variant PCR
Reverse transcriptase-PCR.
Nested-PCR.
Hot-start PCR.
Quantitative PCR.
Multiplex-PCR.
Mutagenesis by PCR.
Allele specific PCR.
…..

Reverse Transcriptase - PCR
RT-PCR, one of the most sensitive methods for

Nested PCR
Nested PCR is a very specific PCR amplification.
Nested PCR use

Hot - Start PCR
Hot Start PCR significantly improves specificity, sensitivity and

Real Time PCR
Traditional PCR has advanced from detection at the end-point

Infectious Diseases/ Cancer
Detection of infectious agents, such as Pathogenic bacteria, Viruses

Genetic Desease
Single point mutations can be detected by modified PCR techniques

Prenatal Diagnosis
Prenatal sexing: Often required in families with inherited sex-linked diseases.
Prenatal

Research
PCR is used in research laboratories in DNA cloning procedures, Southern