Презентация Polymerase chain reaction онлайн
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- Тип файла:ppt / pptx (powerpoint)
- Всего слайдов:54 слайда
- Для класса:1,2,3,4,5,6,7,8,9,10,11
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Слайды и текст к этой презентации:
№6 слайд
![Polymerase Chain Reaction PCR](/documents_6/203e1778ff707773ea28886081bcdd3b/img5.jpg)
Содержание слайда: 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.
№11 слайд
![Primers Typical primers are -](/documents_6/203e1778ff707773ea28886081bcdd3b/img10.jpg)
Содержание слайда: 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
№13 слайд
![The standard PCR buffer](/documents_6/203e1778ff707773ea28886081bcdd3b/img12.jpg)
Содержание слайда: The standard PCR buffer contains:
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
№14 слайд
![DNA Polymerase The most](/documents_6/203e1778ff707773ea28886081bcdd3b/img13.jpg)
Содержание слайда: 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.
№20 слайд
![Sample Handling Use sterile](/documents_6/203e1778ff707773ea28886081bcdd3b/img19.jpg)
Содержание слайда: 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.
№21 слайд
![Laboratory Facilities Set up](/documents_6/203e1778ff707773ea28886081bcdd3b/img20.jpg)
Содержание слайда: 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.
№25 слайд
![Thermal Cycling Profile for](/documents_6/203e1778ff707773ea28886081bcdd3b/img24.jpg)
Содержание слайда: 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°.
№29 слайд
![Number of Cycles The number](/documents_6/203e1778ff707773ea28886081bcdd3b/img28.jpg)
Содержание слайда: 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.
№35 слайд
![PCR Three Phases Exponential](/documents_6/203e1778ff707773ea28886081bcdd3b/img34.jpg)
Содержание слайда: 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.
№39 слайд
![Reverse Transcriptase - PCR](/documents_6/203e1778ff707773ea28886081bcdd3b/img38.jpg)
Содержание слайда: 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.
№43 слайд
![Hot - Start PCR Hot Start PCR](/documents_6/203e1778ff707773ea28886081bcdd3b/img42.jpg)
Содержание слайда: 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.
№45 слайд
![Real Time PCR Traditional PCR](/documents_6/203e1778ff707773ea28886081bcdd3b/img44.jpg)
Содержание слайда: 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.
№48 слайд
![Infectious Diseases Cancer](/documents_6/203e1778ff707773ea28886081bcdd3b/img47.jpg)
Содержание слайда: 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.
№49 слайд
![Genetic Desease Single point](/documents_6/203e1778ff707773ea28886081bcdd3b/img48.jpg)
Содержание слайда: 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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