Types of PCR Real time PCR applications of Real Time PCR

1. Polymerase Chain Reaction (PCR)

2. PCR Recipe Template DNA 50-100ng/μl Reaction buffer (Tris-HCl, ammonium ions, KCl), magnesium ions, bovine serum albumin) This buffer provides the ionic strength and buffering capacity needed during the reaction. MgCl2 - 1.5- 3mM dNTPs -Equimolar ratios, 200 μM each dNTP Primers (0.1 and 0.5 μM) DNA polymerase -1-2 unit/25 μl reaction

3. Variations of the PCR Colony PCR Nested PCR Multiplex PCR AFLP PCR Hot Start PCR In Situ PCR Inverse PCR Asymmetric PCR Long PCR Long Accurate PCR Reverse Transcriptase PCR Allele specific PCR Real time PCR

4. Colony PCR Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

5. Hot Start PCR This is a technique that reduces non-specific amplification during the initial set up stages of the PCR The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase activity at room temperature.

6. Asymmetric PCR Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of one primers for the chosen strand.

7. Nested PCR Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Advantage- Very low probability of nonspecific amplification

10. Ligation of linkers to all restriction fragments.

11. Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.

14. Multiplex PCR Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.

15. In Situ PCR In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells. Applies the methodology of hybridization of the nucleic acids. Allows identification of cellular markers Limited to detection of non-genomic material such as RNA, genes or genomes

16. In Situ PCR

17. Long PCR Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. Application- to clone large genes not possible with conventional PCR.

18. Reverse Transcriptase PCR Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligodT primers (37°C) 1 hr. “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers. Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

19. Allele-specific PCR Used for identify of SNPs. It requires prior knowledge of a DNA sequence, including differences between alleles. Uses primers whose 3' ends encompass the SNP PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer Successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence

20. RealTime-PCR

21. What is Real Time PCR? Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.

22. History of Real Time PCR Initial work by Higuchi and first demonstrated the simultaneous amplification and detection of specific DNA sequences in real-time by simply adding ethidium bromide (EtBr) to the PCR reaction so that the accumulation of PCR product could be visualised at each cycle. (Higuchi et al., 1992) When EtBr is bound to double-stranded DNA and excited by UV light it fluoresces. Kinetic PCR: Continuously measuring the increase in EtBr intensity during amplification with a charge-coupled device camera (Higuchi et al., 1993).

23. Introduction

24. Real Time PCR Introduction General Principles& Concepts What are Fluorescent dyes? Fluorescence Resonance Energy Transfer (FRET) Some commonly used flurophores for labeling probes Quantitating Fluorescence Improving Fluorescence Signal Detection

26. This shift makes it possible to separate excitation light from emission light with the use of optical filters.

28. The efficiency with which it converts absorbed light into emitted fluorescent light (quantum efficiency). Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks.

29. What is Fluorescence Resonance Energy Transfer (FRET)? FRET isa distance dependent interaction between the excited states of2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon

30. A A D (FRET +ve) hv hv hv hv A D R (a) Physical proximity + hv Q FRET (cont’d): The Donor and Acceptor in close physical proximity (10 -100 Angstrom) can lead to FRET or Quenching D (b) No physical proximity + hv (c) No hv Hybridization probes R Q (e) No Physical proximity + hv (d) Physical proximity + hv (Quenching) (Quenching released) TaqMan & Beacon Probes

32. The tight beam of light is sent through a filter which removes most of the light outside of the target wavelength range.

33. The filtered light beam passes through the liquid target sample striking some of the fluorescent molecules in the sample.

34. Light emitted from the fluorescent molecules travels orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range.

37. Real Time PCR Instruments

39. Rotor-Gene (Corbett Research)

40. iCycler (BioRad)

41. Mx4000™ Multiplex Quantitative PCR System

42. ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)

44. Real Time Detection 1a. Excitation filters 1b. Emission filters Tungsten halogen light source (350 - 1000nm continuous) Microplate format Cycler iCycler from BioRad

45. Probe types & Design

47. SYBR Green II

48. EVA Green

49. LC Green

50. BEBO

51. YO-PRO

53. Higher selectivity for dsDNA

54. Lesser sequence dependent

55. Higher stability

56. Lesser inhibitory for Taq

57. Higher resolution in melting curves

61. Probe hybridizes to the target

62. dsDNA-specific 5'—>3' exonuclease activity of Taq or Tth cleaves off the reporter

63. Reporter is separated from the quencher.

64. Fluorescent signal

66. Easy PCR setup

68. Probe design and positioning challenging

69. Similar conditions for primers and probes

70. Elevated background (Quenching capacity)

73. The probe denatures and the loop anneals to the target sequence of the amplicon

74. Separating the quencher from the fluorophore and thereby producing fluorescence which is proportional to the amplicons produced during PCR

75. MB is displaced not destroyed during amplification, because a DNA polymerase lacking 5' exonuclease activity is usedDenaturation Primer molecular Beacon annealing Extension

78. Post PCR analysis

79. PCR multiplex

81. Long probes – less yield

82. Intramolecular competitive binding

84. During the first amplification cycle, the Scorpions primer is extended, and the sequence complementary to the loop sequence is generated.

85. After subsequent denaturation and annealing, the loop of the Scorpions probe hybridizes to the internal target sequence, and the reporter is separated from the quencher. The resulting fluorescent signal is proportional to the amount of amplified product in the sample.

88. During real-time PCR, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur.

90. Hybridization probes h A D h FRET A D A D LC red 640 FAM Probe 2 Probe 1 Amplicon

92. SUNRISE UNIPRIMER PROBE Similar to Molecular Beacon except the stem contains a poly A (15 mer) tail. This tail is complimenatry to the polyT tail of one f the primers. Q AAAAAAAAAAAAAAA PolyA Tail

93. Primer with polyT tail TTTTTTTTTTTTTTT Sunrise Probe with polyA tail binds to the primer polyT tail at annealing. TTTTTTTTTTTTTTT AAAAAAAAAAAAAA Q R hv Q R AAAAAAAAAAAAAA The Sunrise probe changes conformation during denaturation & quenching by DABCYL is removed allowing FITC to fluoresce Sunrise UniPrimer Probe is a modification of Molecular Beacon

94. Commonly Used Fluorescent Probes 78% TaqMan probes 19% Molecular Beacons 15% FRET probes LUX fluorogenic primers 9% 9% MGB Eclipse probes 3% Other 2% Scorpion probes 0% 10% 20% 30% 40% 50% 60% 70% 80% Detection Chemistries

96. Initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline of amplification plot.

97. An increase in fluorescence above the baseline indicates detection of accumulated PCR product.The parameter CT(Threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

98. Effect of Limiting Reagents During the exponential phase, none of the reaction components is limiting; as a result, CT values are very reproducible for reactions with the same starting copy number. On the other hand, the amount of PCR product observed at the end of the reaction is very sensitive to slight variations in reaction components.

100. The threshold line is set in the exponential phase of the amplification for the most accurate reading.

101. The cycle at which the sample reaches this level is called the Cycle Threshold, CT.

102. CT value of 40 or more means no amplification and cannot be included in the calculations.

104. Rn- is the Rn value detected in NTC (baseline value)

105. DRn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR

107. Correlation coefficient (R2) The correlation coefficient is a measure of accuracy of standard curve. Ideally, R2 = 1, although 0.999 is generally the maximum value. Efficiency A PCR efficiency of 100% corresponds to a slope of –3.32. Ideally, the efficiency (E) of a PCR reaction should be 100% but experimental factors such as the length, secondary structure, and GC content of the amplicon can influence efficiency

108. Melt Curve Analysis A melting curve charts the change in fluorescence observed when dsDNA with incorporated dye molecules “melts”, into ssDNA as the temperature of the reaction is raised. when double-stranded DNA bound with SYBR Green I dye is heated, a sudden decrease in fl uorescence is detected when the melting point (Tm) is reached. The fluorescence is plotted against temperature, and then the –ΔF/ΔT (change in fluorescence/change in temperature) is plotted against temperature to obtain a clear view of the melting dynamics.

109. Melt Curve Analysis The probe-based technique is sensitive enough to detect SNP and can distinguish between homozygouswild type, heterozygous and homozygous mutant alleles by virtue of the dissociation patterns produced.

110. Melt Curve Analysis Example: Presence of Primer Dimers

112. The standard curve is based on a serial dilution of a sample with known copy number

113. Ct of each standard sample is plotted against the logarithm of the known concentration

115. Relative Quantification Housekeeping gene: Abundantly and constantly expressed gene. Expression level of these genes remains constant. eg 18 S rRNA, GAPDH, β Actin Normalization: To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples.

117. The Ct values of both the calibrator and the samples are normalized to an endogenous housekeeping gene.

118. This give ∆Ct value of control and the sample.

119. The comparative Ct method is also known as 2-∆∆Ct method, where ∆∆Ct = ∆Ct,sample - ∆Ct,reference

120. Fold change = Efficiency-∆∆Ct or 2-∆∆Ct (which gives relative gene expression)

121. REALTIME-PCRAPPLICATIONS

123. Gene expression

124. viral quantitation

125. Single Nucleotide Polymorphism (SNP) analysis

126. Clinical oncology

127. Cancer

128. Analysis of cellular immune response in peripheral blood

130. SNP Genotyping Multiplex TaqMan assay for SNP genotyping

131. SNP Identification Molecular Beacons

133. LCGreenR, LCGreenRPlus+ (Idaho Technologies)

134. EvaGreen™ dye (Biotium Inc.)