By Dr. Zainab khalid
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1 By Dr. Zainab khalid
2 Introduction to PCR PCR was invented in 1984 by ( Kary mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention
3 What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro to get millions of copies.
4 Why Polymerase? It is called polymerase because the only enzyme used in this reaction is DNA polymerase.
5 PCR tube THERMOCYCLER
6 PCR principle : It allow asingle DNA sequence to be copied (million of times). PCR of two main types: RT_PCR for amplification of RNA. RT_PCR quantitative measurement of RNA or DNA: _The amplification of DNA occurs by repeated cycles of 3 different temp. depended steps: 1-Denaturation:separation of the two strands of DNA by heating up to 94c for 2 min. without breaking the major bonds of its chain.producing single stranded DNA template For replication by thermostable DNA polymerase.
7 2-Anneling: In this step Primers are attached to the single DNAstrand template at their complementary regions, in this step temperature is reduced to 40-60c. 3-Extention: the DNA polymerase synthesize complementary strand. The enzyme reads the opposing strand sequence & extends the primers by adding nucleotides. The temp. in the range 70-74c
8 Denature (heat to 94 o C) Anneling-Lower temperature to o C Extention-Increase temperature to70-74 o C DNA polymerase
9
10 DNA copies DNA copies vs Cycle number Cycle number
11 Amplifies target sequences & increases sensitivity. 1. Ribosomal DNA/RNA. Highly sensitive. 2. Specific sequences of genomic DNA. Highly specific for single species. 3. Random primer amplification Very highly sensitive.
12 Advantages: Small amount of the sample. Very sensitive. + rapid & time consuming test.
13 Disadvantages: Expensive. PCR can fail: - Contamination & false positives. Can not differentiate between living & dead parasite since PCR detect DNA that may still in the body even after treatment & elimination of the parasite.
14 RT_PCR quantitative measurement of RNA or DNA: there will be detection of amplification associated florescence in each cycle. It is used in diagnosis of many conditions other than parasitological infections.
15 Parasites can be diagnosed by PCR : 1- Malaria : - Amplification of oligonucleotide primer pairs within the ribosomal RNA genes which is specific to each species by the Polymerase Chain Reaction (PCR), to detect each malaria species.
16 Traditional diagnosis of Malaria
17 2- Leishmania donovani Amplification of xeno-mini gene found uniqu on it,s genome in bone morrow or tissue sample.
18 3- Toxoplasma gondii Toxoplasma gondii has a three (I, II, and III) strains according to genetic structure. Type II strains cause most cases of symptomatic human infections.this strains can be detected by amplification of DNAof the parasite in blood sample or amniotic fluid by PCR.
19 4-Trypanosoma cruzi Dgnosis of acute infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease by amplification of DNA in blood sample give earlear diagnosis than traditional methods by 3-9 days.
20 3- E. histolytica/e. dispar, G. lamblia and C. parvum/c. hominis infections Their diagnostis by PCR is very limited by *time-consuming methods for the isolation of DNA from faecal specimens * the presence of inhibitory substances in such samples.
21 But it is usful in the epidemiological studies to collect data on the prevalence of E. histolytica and E. dispar which is more advantageous than the ELISA by: time-consuming sensitive Can detect five cysts in the stool sample can be performed in one day and selectively differentiates E. histolytica from E. dispar DNA.
22 Conclusion: The speed and ease sensitivity & specificity of PCR has made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.
23
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