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Презентация на тему Polymerase chain reaction

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2. POLYMERASE CHAIN REACTION
3. ContentsPolymerase Chain ReactionPCR Reaction ComponentsStandard PCR ReactionAvoiding
5.  Coping Machine for DNA Molecule Invented by Kary Mullis and his colleagues in the 1983
6. Polymerase Chain ReactionPCR: Technique for in vitro
7. PCR
8. PCR reaction componentsшаблонA, G, C, TMg2+(forward and reverse)
9. PCR reaction componentsDNA templateTwo primersFour normal deoxynucleosides triphosphatesBuffer systemDNA polymerase I
10. DNA TemplateIntegrityHigh molecular weightPurityPureAmountHuman genomic DNA should be up to 500ngBacterial DNA 1-10ngPlasmid DNA 0.1-1ng
11. PrimersTypical primers are 18-28 bases in length,Having
12. Four Normal Deoxynucleosides TriphosphateFinal concentration of dNTPs
13. Tris-HCl 10mM (10-50mM) for
14. DNA PolymeraseThe most widely characterized polymerase is
15. Enhance The Specificity and or Efficiency of
16. Calculation of Melting TemperatureTm= 2 C° X
17. STANDARD PCR REACTION
18. PCR
19. AVOIDING CONTAMINATION
20. Sample HandlingUse sterile techniques and always wear
21. Laboratory FacilitiesSet up physically separated working places
22. Working with RNA Do not touch a
23. Polymerase Chain Reaction
25. Thermal Cycling Profile for Standard PCR Initial
26. Each cycle includes three successive steps:
27. PCR
28. Exponential AmplificationAs amplification proceeds, the DNA sequence
29. Number of CyclesThe number of cycles required
30. GEL ELECTROPHORESIS
31. Agarose Gel ElectrophoresisIt is a method used
32. Gel Tray/ Loading
33. PCR ProductDNA Molecular Marker Amplified fragments can
34. » Factors, affect the mobility of molecules in gel ChargeSizeShapeBuffer conditionsGel concentration andVoltage
35. PCR: Three PhasesExponential: Exact doubling of product
36. PCR Phases
37. Polymerase Chain ReactionAdvantages of PCRUseful non- invasive
38. Variant PCRReverse transcriptase-PCR.Nested-PCR.Hot-start PCR.Quantitative PCR.Multiplex-PCR.Mutagenesis by PCR.Allele specific PCR.…..
39. Reverse Transcriptase - PCRRT-PCR, one of the
40. RT- PCR
41. Nested PCRNested PCR is a very specific
42. Nested - PCR
43. Hot - Start PCRHot Start PCR significantly
44. Hot - Start PCR
45. Real Time PCRTraditional PCR has advanced from
46. Real Time PCR
48. Infectious Diseases/ CancerDetection of infectious agents, such
49. Genetic DeseaseSingle point mutations can be detected
51. Prenatal DiagnosisPrenatal sexing: Often required in families
52. ResearchPCR is used in research laboratories in
53. Polymerase Chain Reaction
54. Скачать презентацию
55. Похожие презентации

POLYMERASE CHAIN REACTION

 Coping Machine for DNA Molecule Invented by Kary Mullis and his colleagues in the 1983

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

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 DNA 1-10ngPlasmid DNA 0.1-1ng

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

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

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

DNA PolymeraseThe most widely characterized polymerase is that from Thermus aquaticus (Taq),

Enhance The Specificity and or Efficiency of a PCRBetadine

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

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

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

Working with RNA Do not touch a surface after putting the gloves

Each cycle includes three successive steps:

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

Number of CyclesThe number of cycles required for optimum amplification varies depending

Agarose Gel ElectrophoresisIt is a method used in biochemistry and molecular biology

PCR ProductDNA Molecular Marker Amplified fragments can be visualized easily following staining

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

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

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

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 the

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

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

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

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

Genetic DeseaseSingle point mutations can be detected by modified PCR techniques such

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

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

Слайды презентации

Слайд 2 POLYMERASE CHAIN REACTION

Слайд 3 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

Слайд 4

Слайд 5  Coping Machine for DNA Molecule
 Invented by

Kary Mullis and his colleagues in the 1983

Слайд 6 Polymerase Chain Reaction
PCR: Technique for in vitro (test

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

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.

Слайд 7 PCR

Слайд 8 PCR reaction components
шаблон
A, G, C, T
Mg2+
(forward
and reverse)

Слайд 9 PCR reaction components
DNA template
Two primers
Four normal deoxynucleosides triphosphates
Buffer

system
DNA polymerase I

Слайд 10 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

Слайд 11 Primers
Typical primers are 18-28 bases in length,
Having 40-

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

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

Слайд 12 Four Normal Deoxynucleosides Triphosphate
Final concentration of dNTPs should

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

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.

Слайд 13 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

Слайд 14 DNA Polymerase
The most widely characterized polymerase is that

DNA PolymeraseThe most widely characterized polymerase is that from Thermus aquaticus

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.

Слайд 15 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,….

Слайд 16 Calculation of Melting Temperature
Tm= 2 C° X (number

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

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 .

Слайд 17 STANDARD PCR REACTION

Слайд 18 PCR

Слайд 19 AVOIDING CONTAMINATION

Слайд 20 Sample Handling
Use sterile techniques and always wear fresh

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

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.

Слайд 21 Laboratory Facilities
Set up physically separated working places for:
Template

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

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.

Слайд 22 Working with RNA
Do not touch a surface

Working with RNA Do not touch a surface after putting the

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.

Слайд 23 Polymerase Chain Reaction

Слайд 24

Слайд 25 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°.

Слайд 26 Each cycle includes three successive steps:

Слайд 27 PCR

Слайд 28 Exponential Amplification
As amplification proceeds, the DNA sequence between

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

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.

Слайд 29 Number of Cycles
The number of cycles required for

Number of CyclesThe number of cycles required for optimum amplification varies

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.

Слайд 30 GEL ELECTROPHORESIS

Слайд 31 Agarose Gel Electrophoresis
It is a method used in

Agarose Gel ElectrophoresisIt is a method used in biochemistry and molecular

biochemistry and molecular biology to separate DNA, or RNA

molecules based upon charge, size and shape.

Agarose is a polysaccharide derivative of agar.

Слайд 32 Gel Tray/ Loading

Слайд 33 PCR Product
DNA Molecular Marker
Amplified fragments can be

PCR ProductDNA Molecular Marker Amplified fragments can be visualized easily following

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

Слайд 34 » Factors, affect the mobility of molecules in

gel
Charge
Size
Shape
Buffer conditions
Gel concentration and
Voltage

Слайд 35 PCR: Three Phases
Exponential: Exact doubling of product is

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

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.

Слайд 36 PCR Phases

Слайд 37 Polymerase Chain Reaction
Advantages of PCR
Useful non- invasive procedure.
Simplicity

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

of the procedure.
Sensitivity of the PCR

Disadvantages of PCR
False positive

results (cross contamination).
False negative results

Слайд 38 Variant PCR
Reverse transcriptase-PCR.
Nested-PCR.
Hot-start PCR.
Quantitative PCR.
Multiplex-PCR.
Mutagenesis by PCR.
Allele specific

PCR.
…..

Слайд 39 Reverse Transcriptase - PCR
RT-PCR, one of the most

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

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.

Слайд 40 RT- PCR

Слайд 41 Nested PCR
Nested PCR is a very specific PCR

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

amplification.

Nested PCR use two pairs (instead of one pair)

of PCR primers are used to amplify a fragment.

Слайд 42 Nested - PCR

Слайд 43 Hot - Start PCR
Hot Start PCR significantly improves

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

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.

Слайд 44 Hot - Start PCR

Слайд 45 Real Time PCR
Traditional PCR has advanced from detection

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

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.

Слайд 46 Real Time PCR

Слайд 47

Слайд 48 Infectious Diseases/ Cancer
Detection of infectious agents, such as

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

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.

Слайд 49 Genetic Desease
Single point mutations can be detected by

Genetic DeseaseSingle point mutations can be detected by modified PCR techniques

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.