المحاضرة 6 - فايروسات عملي

Polymerase Chain Reaction (PCR)

PCR, polymerase chain reaction, is an in-vitro technique for (amplification of specific region of DNA) whose sequence is known or which lies between two regions of known sequence

Before PCR, DNA of interest could only be amplified by

 over-expression in cells and this with limited yield

Reaction Components

1. DNA template

2. Oligonucleotide Primers

3. Enzyme

4.=  dNTPs

Deoxynucleoside triphosphate synthesis = DNTPS

5. MgCL affects primer annealing and Taq activity

6. buffers

7. Working concentrations

8. KCL (10-50 mm)

9. Tris-HCl (10 mM, pH 8.3)

10 NaCl (sometimes)

يعني باختصار ( K+H+Na * CL )

The PCR Cycle

Comprised of 3 steps: 

1. Denaturation of DNA at 95C
2. Primer hybridization (annealing) at 40-59°C - تذكر مرام ( نيالكم احنا ما عدنا )
3. DNA synthesis (Primer extension) at 72°C

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PCR Cycle - Step 1

- Denaturation Template DNA by Heat (95°C

PCR Cycle - Step 2

Temperature is lowered (T) and primers anneal to target

sequences 

PCR Cycle - Step 3

At 72°C Tag DNA polymerase catalyses primer extension as complementary mucleotides are incorporated

End of the 1st PCR Cycle

Results in two copies of target sequence

Target Sequence

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Standard thermocycler

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Reverse transcription polymerase chain reaction (RT-PCR)

• . It is one of many variants of polymerase chain reaction.
•  It was introduced in 1977.
•  The discovery of reverse transcriptase during the study of viral replication of genetic material led to the development of RT-PCR RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA (CDNA) transcripts from RNA. So, first step of

يوجد تقنيتين مرتبطتين بـ pcr

1. reverse transcriptase = RT - PCR 
2. real time = RT - PCR 

RT PCR is

1- isolate the mRNA from the cell

2- Next, make DNA from the mRNA This is reversing "transcription" so use an enzyme originally

obtained from viruses enzyme IS called reverse transcriptase

(The RT of RT PCR) 

الانزيم الذي يحول الـ RNA الى DNA يسمى reverse transcriptase

 

• This technique is commonly used in molecular biology to detect RNA expression .

RT-PCR principle

Traditionally, RT-PCR involves two steps: the RT reaction and PCR amplification.

الخطوة الاولى : Reaction

1.  RNA is first reverse transcribed into complementary DNA (CDNA) using an enzyme reverse transcriptase 

After RT, you now have a tiny, trace amount of what is called complimentary DNA (CDNA). This tiny trace amount is   to not enough sequence

• Complimentary DNA = (CDNA)
• CDNA ≠ DNA 

الخطوة الثانية : amplification

2. The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes

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