Different types of PCR and principles of Real Time PCR. Prof. Dr. Hamdy M. El-Aref Assiut University, Faculty of Agriculture Genetics Department
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1 Different types of PC and principles of eal Time PC. Prof. Dr. Hamdy M. El-Aref Assiut University, Faculty of Agriculture Genetics Department
2 I N T O D U C T I O N PC Cycle (round)
3 I N T O D U C T I O N
4 Types of PC Standard PC, DNA T-PC, NA eal-time PC TQ-PC (DNA or NA)
5 everse-transcriptase transcriptase-polymerase polymerase chain reaction T-PC In T-PC, specific mna could to be detected and quantified. In T-PC, reverse transcriptase (T) is used to copy all of the mnas in an NA sample into cdna. This single stranded cdna can then be amplified by PC using primers that anneal to a specific cdna (vis.. mna).
6 T-PC 5 -Cap mna AAA(A) n (dt) 12~18 primer anneal 3 5 AAA(A) n dntp everse transcriptase cdna:mna hybrid AAA(A) n 5 egular PC
7 T-PC for Gene expression
8 One step T -PC Two steps T and PC
9 What is eal-time PC? detection and the quantification of a fluorescent transmitter during the process of amplification. Can view PC cycles in real time DNA is quantified after each cycle of PC Therefore can simultaneously quantify and amplify Can detect the number of copies in the sample. no post-pc processing of products. (No gel-based analysis at the end of the PC reaction)
10 How to measure the PC product? Two general principles for the quantitative detection of amplicons: 1- agents (dye) binding to the double-stranded stranded- DNA (SybrGreen( I). 2- fluorescent probes (FAM, TAMA, JOE, OX,) * TagMan analysis (Hydrolysis probes ) * Molecular Beacons, * Scorpions TM other probes
11 Initial DNA strand First PC cycle Second PC cycle Third PC cycle Fourth PC cycle
12 Any increase in fluorescence level can be plotted onto a graph and easily interpreted Fluorescence level Virus present Cycles No virus
13 ealtime Fluorescent PC with SYB Green When the fluorescent probe SYB green is present during a PC reaction, it binds to the doublestranded PC product and emits light at 520 nm. The SYB Green dye only fluoresces when bound to DNA. Hence, the amount of fluorescence correlates with the amount of PC product produced. This allows the accumulation of PC product to be followed through many cycles.
14 SYB Green I No probe; lower reagent cost than TaqMan. No more than 0.5 µg of DNA in sample Believed to be a minor groove binding dye.
15 - Economic - Easy to use The SYB Green: advantages - Has more sensibility than the ethidium bromide (another intercalating agent) - Does not inhibit the reaction of amplification - Does not require any fluorescent probe,, thus does not require any particular expertise for the design of the probes - Is not affected by mutations in the target DNA
16 The SYB Green disadvantages Impossible to make sure of specificity of amplicons Bad pairing can lead to positive forgeries or an over-estimate estimate of the quantification Still unspecified mutagen capacity
17 ealtime Fluorescent PC with TaqMan Probe (probe hydrolysis) The TaqMan probe has three elements: a shortwavelength fluorophore on one end (diamond), a sequence that is specific for the target DNA (blue), and alongwavelength fluorophore at the other end (circle). The two fluorophores are so close that fluorescence is quenched and no green light is emitted. This probe is designed to anneal to the center of the target DNA. When Taq polymerase elongates the second strand during PC, its nuclease activity cuts the probe into single nucleotides. This releases the two fluorophores from contact and abolishes quenching. The short-wavelength fluorophore can now fluoresce and a signal will be detected that is proportional to the number of new strands synthesized. quencher Molecule that prevents fluorescence by binding to the fluorophore and absorbing its activation
18 TaqMan Uses a Fluorescent Probe eporter dye Quencher dye
19 Taq Extends from Upstream Primer
20 5 -Nuclease Activity Digests Probe
21 Primer Probe TaqMan SYB Green probe hydrolysis agents binding No probe
22 2- Theory of eal-time PC? TaqMan Chemistry Q
23 2- Theory of eal-time PC? TaqMan Chemistry Q
24 2- Theory of eal-time PC? TaqMan Chemistry Q
25 2- Theory of eal-time PC? TaqMan Chemistry Q
26 2- Theory of eal-time PC? Molecular Beacons Q Q Q Q Q Q
27 2- Theory of eal-time PC? SCOPIONS Q
28 2- Theory of eal-time PC? SCOPIONS Q Q Q Q Q Q Q
29 Published eferences for real-time PC Fluorescent Chemistries No. of references Taqman SYB Green molecular beacons scorpions Year
30 Advantages and Disadvantages Advantages: Fast PC eg 30 cycles in 20 minutes Quick = faster results = faster treatment Increased specificity Qualitative and Quantitative Easy interpretation of results Small sample volume Disadvantages: To respect the principles of design of the probes Expensive
31 30 bases in length Probe design G-C C content of around 50 % The probes should not overlap with, or have sequence complementarity with either of the primers Probes should not contain a G at their 5 5 ends, because such an arrangement quenches reporter fluorescence, even after cleavage A Tm of 5 to 10 C C greater than that of the primers
32 Q T-PC Applications Gene expression. DNA target quantification (nuclear, mitochondrial DNA). SNP detection. Viral load assays, pathogen & GMO detection. Clinical Diagnostics (Cancer, Therapy esponse) cdna cloning (cdna( Library).
33 eferences Micklos, David, Greg Freyer and David Crotty. DNA Science a First Course. New York:Cold Spring Habor Laboratory Press, Purves, Sadava, Orians, Heller. Life. 6th ed. Sinauer Associates, Demidov.V, Broude. N(2004). DNA Amplification: Current Technologies and Applications Websites: (Animation) ( PC Animation)
34 Prof. Prof. Dr. Dr. Hamdy Hamdy El-Aref El-Aref
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