관리자
22 Jul 2021
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Contents

1.Introduction 

2.Materials and equipment 

3. Procedure 

4. Results 

5. Precautions 

1. Introduction


1. Introduction

To understand the mechanism of *real-time PCR by using the DNA of **Chlamydia trachomatis, one of the bacteria that cause sexually transmitted diseases.

* What is real-time PCR? It is also known as quantitative PCR, for its ability to accurately quantify PCR products that are being amplified by real-time monitoring. The amount of DNA increases at a rate of 2ⁿ for every PCR cycle, where n denotes the number of cycles, where the amount is drawn on a graph simultaneously. Because conventional PCR ensures the presence or absence of a band through electrophoretic methods, real-time PCR may be a choice for higher sensitivity and resolution.

** What is Chlamydia trachomatis? Chlamydia trachomatis, commonly known as chlamydia, is a sexually transmitted bacterium that can cause various infections, which can be manifested as trachoma, lymphogranuloma venereum, non-gonococcal urethritis, cervicitis, salpingitis, and pelvic inflammatory diseases.


2. Materials and equipment


2. Materials and equipment 

Materials

TaqMan Gene expression master mix, Primer set (Forward, Reverse, *Probe), Chlamydia trachomatis DNA, dH2O

*What is a probe? In real-time PCR, a primer with fluorescent molecule attached is added to the pair of primer set. The fluorescent molecule does not emit light during the annealing step, but in the extension phase the molecule gets detached from the primer by DNA polymerase, emitting fluorescent light. The amount of DNA can be quantified in real-time by measuring intensity of light emission.


  • Primer information

F: GCGGGCGATTTGCCTTA

R: CGGTCAACGAAGAGGTTTTGTC

  • Probe information

P: CGGAGCGAGTTACG 

Equipment

Real time PCR (ex.QuantStudio5- Thermofisher)

3. Procedure


3. Procedure 

① Prepare ten-fold serial dilutions of Chlamydia trachomatis DNA.


Sample 1Sample 2Sample 3Sample 4Sample 5
DNA2uL2uL of DNA solution from Sample 12uL of DNA solution from Sample 22uL of DNA solution from Sample 32uL of DNA solution from Sample 4
dH2O18uL18uL18uL18uL18uL
Total20uL20uL20uL20uL20uL


② Composition of PCR Sample Reagent


Sample 1 ~ 5NC
Master Mix10uL10uL
Primer Set5uL5uL
CT DNA3uL-
dH2O2uL5uL
Total20uL20uL


③ Choose the setting appropriate for PCR.

StepsTemperatureTime
Cycles
Early Denaturation
50℃
02:00
1
Denaturation
95℃
10:00
1
Annealing
95℃
00:1540
Extension
60℃
01:00

*Final extension and stabilization at 4℃ are unnecessary because graphical results are obtained real-time.

4. Results


4. Results

The amount of DNA expressed can be confirmed by looking at the graph for each sample and obtaining the Ct value. Ct value, or cycle threshold, is the number of cycles at which the fluorescent signal crosses the threshold. It is recognizable from the graph that the Ct value of each sample gets pushed further as it gets more diluted. By making interpretations from such results, it can be used for other purposes, such as analyzing gene expression, detecting virus or other pathogens.

5. Precautions


5. Precautions 

① Avoid exposure to light, because it may interfere with the fluorescence of the probe.

② Transfer the correct amount of DNA with the pipette during sample dilution.


#Chlamydia trachomatis #Real Time PCR #Quantitative PCR





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