Polymerase chain reaction (PCR) is a technique used to amplify a specific DNA sequence. There are several factors to consider when designing primers for PCR, including length of 18-30 nucleotides, melting temperature between 65-75°C, GC content of 40-60%, and avoiding repeats or secondary structures. Real-time PCR uses fluorescent dyes and probes to quantify the amount of DNA produced during each cycle. SYBR Green binds double-stranded DNA produced and fluorescence increases with more DNA, while TaqMan probes have a reporter dye separated from a quencher until the probe is cleaved during PCR. To amplify RNA via PCR, it is first converted to cDNA using reverse transcriptase, then the cDNA

1. Polymerase chain reaction
PCR
By
Dr. Mona albureikan

11. Primer Design Tips & Tools
• In general, a length of 18–30 nucleotides for primers is good.
• Try to make the melting temperature (Tm) of the primers
between 65°C and 75°C.
• If the Tm of your primer is very low, try to find a sequence with
more GC content, or extend the length of the primer a little.
• Aim for the GC content to be between 40 and 60%, with the 3'
of a primer ending in C or G to promote binding.
• Typically, 3 to 4 nucleotides are added 5’ of the restriction
enzyme site in the primer to allow for efficient cutting.
• Try to avoid regions of secondary structure, and have a
balanced distribution of GC-rich and AT-rich domains.
• Try to avoid runs of 4 or more of one base, or dinucleotide
repeats (for example, ACCCC or ATATATAT).
• The 5’ (forward) primer must contain a start codon (ATG).

12. Example
Coding: 5’-ATGGCAGAGATGGGCAGTAAA.other stuff. ACTTCACTGAGAGGGTCCCATGA-3’
Complementary: 3’-TACCGTCTCTACCCGTCATTT .other stuff. TGAAGTGACTCTCCCAGGGTACT-5’
To design the forward primer
5’- ATG GCA GAG ATG GGC AGT AAA
For the reverse primer
5’- TGG GAC CCT CTC AGT GAA GTT

13. Real-Time PCR (TaqMan Method)
1- Reporter (Green Fluorescent Protein (GFP).
2- Quencher (Red Fluorescent Protein (RFP).
- The reporter dye is found on the 5’ end of the probe and the
quencher at the 3’ end.
- The probe consists of fluorescent parts;

14. While the probe is attached or unattached to the template DNA and
before the polymerase acts, the quencher (Q) fluorophore (usually a
long-wavelength colored dye, such as red) reduces the fluorescence
from the reporter (R) fluorophore (usually a short-wavelength
colored dye, such as green). It does this by the use of Fluorescence
,which is the inhibition he inhibition of one dye caused by another
without emission of a proton.
Real-Time PCR (TaqMan Method)

15. -Removes the Taqman® probe from the template DNA.
- This separates the quencher from the reporter, and allows the
reporter to give off its emit its energy.
- This is then quantified using a computer.
Real-Time PCR (TaqMan Method)

16. SYBR Green
- SYBR Green can’t bind single stranded DNA or primer.

17. - But it can bind double stranded DNA once the polymerase has
made the 2nd strand.
- The amount of fluorescence is proportional to the (log) amount
of ds DNA.
SYBR Green

18. PCR of RNA

19. PCR of RNA

20. • Then do normal PCR of the cDNA.
• This is called RT-PCR (Reverse transcription polymerase chain
reaction (RT-PCR)
Can we “amplify” RNA?
– The enzyme that carries out the PCR
reaction is a DNA polymerase
– SO….it doesn’t DO RNA
– AND its not very quantitative
PCR of RNA
• First – need to REVERSE
TRANSCRIBE the RNA
• An enzyme called reverse
transcriptase is used to copy the RNA
back into complementary DNA (cDNA)
PCR of RNA

21. References
• Deferent si- The informations and pictures from different
sits.