A lecture by real-time PCR writen by iqra Rasheed

1. Real-time PCR
iqrarashidgcfw@gmail.com

2. Road Map
 What is PCR?
 Components of PCR
 Steps of PCR
 Real time PCR
 Steps of real-time PCR
 Types of Real-time PCR
 SYBR green and Taqman probe
 Methods of real-time PCR
 One-step vs. two-steps real-time PCR
 Applications of real time PCR

3. PCR (polymerase chain reaction)
 Kerry Mullis 1985
 In vitro DNA Replication
 DNA sequence to be selectively amplified
 Smaller sample of DNA to be cloned
 Millions of copies in just a few hours

4. Components of PCR Master mix
 DNA template
 Oligonucleotide primers
 DNA Taq polymerase
 dNTPs
 PCR Buffer

5. Steps of PCR
 Denaturation
o Double stranded DNA is denatured to single strand
o Breaks H bonding
 Annealing
o Temperature is rapidly lowered
o Primer binding
 Elongation
o Polymerase enzyme sequentially adds bases to the 3′
each primer
o 5′ to 3′ extension

7. Cyclic reaction
 One cycle, single segment of DNA template is
amplified into two separate pieces of double stranded
DNA
 Two pieces are then available for amplification in the
next cycle
 Cycle is repeated
 More and more copies are generated

9. Real-time PCR
 PCR product is measured at each cycle
 Doubling the number of target molecules with each
amplification cycle
 Fluorescent dyes that yield increasing fluorescent
signal in direct proportion to the number of PCR
product molecules (amplicons) generated

11. Advantages of real-time PCR
 Ability to monitor the progress of the PCR reaction as it
occurs in real-time
 Measure the amount of amplicon at each cycle
 Quantified the amount of starting material in samples
 Amplification and detection occurs in a single tube
 Use to measure gene expression

12. Steps of Real-time PCR
 Conversion of RNA to cDNA (reverse transcription)
 Uses Fluorescent reporters
 Two types of fluorescent reporters are used
 SYBER green dye
 Taqman probe

13. SYBER green dye
 Bound to double stranded DNA
 Dye binds minor groove of all DNA
 More product more dye bound
 Cheap
 Non specific

14. Taqman probe
 Dye and quencher either end of the probe
 Probe binds to the DNA between the forward and
reverse primer
 Quencher absorbs the fluorescence emitted by the
reporter
 During the extension phase of the PCR reaction the
probe is degraded
 Releasing the reporter and allowing its fluorescence to
be detected

16. Methods of Real-time PCR
One step real-time PCR
 Certainly easier to set up with less overall hands-on time
 Reverse transcriptase step in the same tube as the PCR
reaction
 Advantages
 Fewer pipetting steps
 Best method when only a few assays are run repeatedly
 Multiplex PCR of gene of interest and control can be
done in single well, from same RNA sample

17. Two steps real-time PCR
 The second method involves creating cDNA first by
means of separating reverse transcription reaction and
then adding the cDNA to the PCR reaction (two-step)
 Advantages
 Highly sensitive
 Potentially more efficient because random primers and
oligo d(T) can be used
 Possibility to stock cDNA to quantify several targets
 Recommended when the reaction is performed with a
limiting amount of starting material

19. Application of real-time PCR
 Real-Time PCR has become a cornerstone of molecular
biology Just some of the uses include:
 Gene expression analysis
 Cancer and Drug research
 Disease diagnosis and management
 Viral quantification
 Food testing
 Percent GM food
 Animal and plant breeding
 Gene copy number
 Forensics
 Sample identification and quantification

20. Sources
 https://genome.cshlp.org/content/6/10/986.short
 https://www.nature.com/articles/6364190
 https://scholar.google.com/scholar?hl=en&as_sdt=0%2C
5&q=real+time+pcr&btnG=
 https://www.bioline.com/one-step-vs-two-step-real-time-
pcr.html